memorize

cell funadment unitl there is bactieral disorganized, mized, cytoplasn, organels, nucleus, dna all jumbled - then there are mammailan cells that are super oganized and seprated. there ar 210 cells and all ahve smae information but they all come sfrom the ferilzied egg. and it is differfentaion and proliferation that makes these cells different abd hav th same genetic ifromation b ecaus eidfferent cells are activated with different gnes, tim period, enviorment. so the cell has a specific window where it can take in singals and differente and refplciate. once its done this is called post mitotic. 

homolgous genes devlopment in diveres animals - house keeping gens are gesne are expressed in all cells to aminitn suvivel. 

homoglous genes mean that they come from somon ancestor. homologu sgens means they coem from a common ancestor. then there are fuck ass compoudn eyes - it is a eye mad eup of hudnreds of lenses in a fly calle domatiidim . ommitidium is a lense that makes of hundreds and ths is a compound eye and it has the gene called eyeless and without it the the fly has no eyeaga…

homoelgous genes means that they coem from a common ancesotry. homoglous homoglous genes - commonancestory. then they hav compodun eyes and they are mad eup of hundreds of lesnes called ommitidum and htey need the gene called yeeles ucz without it then there is no eyes. tis is ismilar to the geen for humans called pax six that wthout it the human has no irirs. lolol but it is a heterozygous eltion becuase if it were homozoguys then hte organism wouldnt be able to ausuve. the compound uee= -ooomaitim lenses. 

the cell si the funcamental unit. the cell cultuer celled is a population of homgenosus. media dnex periemtnal contridiotn - it is so the cells can grown multiple closer to human reslbled sa tisse, can b eused as a model sytstem not tehe best tho we still need lvie stuff. 

so the cell will recive ingal that are specific to it and and psieicf to its receptors. it can iether go to its long term or quick response . quick is jus tmoifying ecisisting gproteins to transmit the singal and long term s i making ne wpreotisn. okie so basicallyh te cells have a speicif tim eperiod before they can differentation and take in singals and all of that good stuff. okie so basically stem cells or embyonic cells have a speicif itme period to differenitatek and then go to their respective body region o form organs or what not but once that window closes it is done. cancer cells will not work with that process and will divide and travel and beocme whatver they want and it leads to mestaizing. the most dangerous part isnt the turm it is mestatize bcause if it travels it is harder to control. --

the most abbundant class of phospholipid in animal cell membranes, with a hydrolphobic taill composed of two fatty acyl chains that are esterified to the two hdryoxyl groups in glcyerol phophaote and a polar head attached to a pshophate group is called

phosphoglcyer - polar heat, phospohate, glycerol, fatty acid group

generally the psohoglucerides are _molcules

amipathic. 

the exoplasmic face of the plasma membraie is directed … away from the cytosol 

the membrane is made up of lipid- phosholipids - phosphoglycerideds are the most common and they are the oens that make up the plasma membrane. they have a phsohopahtoe, glyerol, fatty aceyl tail, and a charged group. they are amiphaothic. there are many tyoes like phoshoadtylserine whichi sin the cytosplamic side and the exoplasmic has phosoadtylchlorine. phosholipids have fatty acids which contribute to the membrane fluidity/viscotiy so basically faty acids with single chains are called saturated and they are strong and make it gel like espeically if it s more longer. then theres the unstatured which prodivde kinks and if theyre shorter then it gives more space and makes it more fluid like which is what we want. it gives it more space and weakens out the vanderwaal interactions. phsohlolipid the fatty acid part are held together y eweak hydrophonic and van der waal interactions. so the unstarued kinks weaken them. these are not convaletn attached. cholestorl is heavily embeeded into the memrbane whic is made up of fatty acids and a charged oH group which also makes it amiphathic. but what increase the visotiy of the whole thing is increasing the temprfature and the more momvnet. viscotity is important becaus eosmtiems the receptors and the memrbane speicif parts have to interact or recpeot and rercpeotr hav eto interact but they dependo on the viscotiy in order to get closer. okie somore about phsoadtyelserine, it works with a receptor to allow a signal to get transmitted in, its a sensory and prvides stability , it is located in the cytosolic leaflet. pshoatdyeserine is a senosro or tramistter of singling events. the distrbituion of the PS ont he cell is important because it will determine if the cell dies or not . so if the cell is unwellthen flipase will hydrolyze atp and then flip it adn then use ti to shove it ot he exoplasmi cisde like in the blood cells and in the blood sream macrophages will be around to engulf it or phagocyte them and destroy them. but this is because there is diroganization of the palsma membrane. this isnt alwaus a indcator that a cell will die becaus sometimes they will flip when there is a cut and there needs to be scabbing. oh i forgot to say that the lipids will move laterally and rotate around. 

SO FRAP strands for flourscen recepvoe after bleaching. flourscent recovery after bleaching. FRAP FRAP like a frapuccino. FRAP is an expeiremt to dtermein the fluidity of the cell membrane. we take a live cell,add cloring ot the proteinsof the cell membran using blue due and then we will use a lazer to quenc the color of an area and then we observe in order to determin the fluidity and to see if the color comes back in that speicifc area and we can measure mobility percentage. 

so we got four differnet types of proteins - transmmebrane - lipid anchor- and peripheral actall my bad jsut three. so basically foru transmembrane they have the cytosolic domain, the exoplasmic domain, and the transmmebrane domeina. and the ligand will recogvnize the exoplasmid domain or something the amino acids of the transmmebrean domina, and the amino acids side chains will interact iwth the hydrophobic fatty acid chains that will inteact through hydrphobic and vanderwaal inkeracitons. anywhoo so the periphal will sit on top and either interact with the membrane inside otuside receptors or lipid heads. they aer philic tranmembreanes amiphathic. then we got anchorlipid proteis .oh i forgot to mentiaon that glycophorin is a homodimer protein transmembrane. tranmembre proteins homodimers like glycophorin a.  the coiled coil dimer stablixed by van der wall interactions. the a helix. okie noug about that we got lipid anchors which are attached through covalent ineractions with the lipid groups . they are imebdedded and have a philic head. we got the prenylation and acetylation groups that are done throug post trancirpiton. prenylation means that a lipid is attached to a protein at the c terminal end and aceyl ation is the n terminal . again this is all don e through post tranlational modification. 

lecture 2 - questions

- a ligand binds to a speicif recepotr atha binds in activation of a singal tranductio pathway .. 

single pass trasmebean passes the ligand once . okie so prenylation happens when a fatty acyl chain will attach to a c terminal . acetylation happens when the protein attaches to the n terminal. so for prenylation what happens is that the protein itself will have a caax motif region whichn is the last four amino acids. they have cystein, val, leu or ala which are allipathic(hydrophobic) and the x is any ranom. so a prenyl group like geranyl or farnesyl will attach to the protein’s cystein thorugh farneysl transferase or a prenyl tranfease and this will be through covalent linkage and make a thioether bond. and then a caax protease will break the last three amino acids and make a negative charge, but then methyl tranferase will add a ch3 group and neutrlize the negative. immediatly the newly made lipid anchor will run and attach to the hydrophoc part of the lipids and attach hydrophoboci and vanderwaal. the negative neutralization is bc it helps the lipid anchor structure go into the lipid transmbreane . its for support and make it smoother. now the lipid anchor can allso attach with the help of. a carbohodyrate intermiedate like GI anchor. 

a way that a cell responds to these singaling events is through singal tranduction process. the recpeots will recognize thoes singling moclules called ligand. singling recpetors meaning either surface receptors or intracellular ecpetors that are in the inside. so there are singling receptrs either in the inside which are intracellula and take in ligands that are hydrophobic and small , and surface receptors which are for hydrophobic ligands. intra cellular could also mean inside the cell. when a recpetor binds ligan it acitvate and there is a conformation change, surface receptors are transmembreane. sometimes the ligand will rexonigize the exoplasmic domain or the transmembrane domain. so when a ligand bind it causes a confirmation change and this increases the affinity for a downstream singling molcule to bind. without the confrimation change then the affinity is much lower. 

ligands can be proteins, peptides, amino acids, nucletoides, steroids, faty acids, and gasses secreted by exocytosis …

part of the response of a cell to this singling event is haveing cellular process hcnage, like the metabolism, the expression, like long term or short and fst, and reuslts in trancirptio factors turnign on or off. the lock and key mecahnism is that the molecular ocmplentary of ligan and receptor. then ther is the moelcular switch which till the ligand binds the receptor is off and like a switch when its one it iwll turn on. post translational modifiation of a protein like phsohroylat, aceylation and others can chang the conromation of a protein and make it on or off. 

there are four types of singals, there is endocrine in which the endocrine cell releasing hromones or singals into the bloodstream to reach different parts of the body , because of this this has a long half life in order to last and have time to bind. then there is paracine which is not touching but in shorter proximity to the reciving cell because the half life is a lot shorter and needs to be fast. so there is a concentration gradient that builds up and then releases in order to transmit the singal. then there is autocrine which invovles the singals being transmitting coming out of a cell and then attaching to the same cells receptors and doing it again. this  can be for proliferation. it stops in normal cells but for other cells like cancer celsl it doesnt stop and keeps growing and dividing and it can go to other areas of other cancer cells and make them divide to. there are four global reponses to singals - survive, die, differentation, and grow and divide. oh i forgot another type that is contact depending, the ligand is bound to the membrane and the cells are phycally touching eeachother, the medium fluid there is non

the more receptors present the better respond, there are diffeent combination that can cause four different tupes of singals, some can cause survival, prolferation and grow, and differentiation, and death if there are no signals. 

a ligand usually binds to a specific  that results in the acivation of a singal tranduction pathway - receptor

in paracien singling the singling molcule - acts on cells in close proximito to the secreting cell

the super family of preoitsn that RAS belongs to is gtpase. 

name the family of preotins by pshucally unteractin ras enhances the intrisic gtpas of rase in the rkt mediaed singal tranduction pathway - GAP

RECEPTORS - 

there is the

NUCLEAR recpetor super family - intracellular hormone receptors

Cell surface receptors - ion channel - g protein coupled, enzyme coupled - transmembrane

so about enzyme couple receptors - they are homodimers like RTK which is a kinase that phrooylates its a trans autophohroylator which does it to itself. so then we got ras which is a gtpase. so when ras is bounded to gtp it is acitve but it isnst the best gtpas so therefore gap will bind and make it faster and the faster it works then the faster it releases that gamma phoshophate and becomes gdp which is the inactive from. we cant turn gdp back to gtp so GEF will come in and will work to remove the gdp to allow a gtp to bind which luckily there is an abundant amount. okie so kinase is a enzyme coupled receptor . ras is a intracellular protien 

ras pathwya sinvolves the recepto RTK which is a tyrosin kinase. ras is not a recpetor but it is a protein, intracellular protien. it is isnde the cell and lipid anchor, RAS is a intracelular gtpas anchor protein. ras is phsycially attached to the membrane like a prenylated protein. a growth factor would nerver grow with a ras. so before the ligand binds to the RTK it has low kinase activity. wron g time and wrong place wat makes ras dengour in cnacer. ras is an enzyme ras is not a kinase. ras changes cofnriamtion through gap and gef. GAP and GEF work to do a confrimation change. RTk is a coupeled recepto rhta tacivates this long term resposne - the recept is a tyroskin kinase that phrohoyalted the tryosiden resudes amino acids. this is needed for cellular porliferiaont and fifrentation. cnacner cells can highkacl the system and use the tyrosine receptors for its benefits and causes increase in proliferation. 

RKT can bind to a ligan like EGF or PDGF

So rtk is a homodimer that will bind to eachother once the ligand binds. It will trans autotranfphrohoylate eachother and change ocnriamtion where GRB2 will recognize it and become and active molecule. then it will recruit sos and sos acts like gef in which is will remove gdp from inactive ras. Binds to ras (which is a intracellular , anchor lipid, gtpase, preynlated moclule) and then it has high affinity for raf which is inactive from 14-3-3 binded to it. once binded it will relase and raf is a kinase and phrohylate mek and that prhooylates mapk and then goes into the nucleus and phrohoylates trancription factors. those genes turn on and will make the cell differentate or proliferate. 

there are hundreds of ttyroskin kinases in the body and not every kinase can phnrohoylate everyprotein. kinases recognize amino acids they posphorylate itll be lik 6-8. even if two proteins have tyrosin the surround amino acids determin ewhich kianse can prhoyalte it. if there was one kinase for alll tyrosin it woul turn on eveyrthing and be choatic. mutliepl kinases controls different pathways. phosphoatases acts as off switches to reset the system. 

so now if the trandcution pathway wants to be turned off this is done through phosphatease coming in and dephoshporylating all kianses like, the rtk receptor, raf, mek, mapk. it can add the 14-3-3- to the raf, and it can add gap to ras-gtp to make it inactive faster. 

so basically grb2 is a mediator because the sh2 domain will recognize the phrohoylated residue of the revptor of trysoisn and recognzie the confirmational change in order to bind and the sh3 is the connector between source and source enzyme whihc is the sos. 

so ras is an oncogene for many tumors because it keeps it on for a hyperactivly. this is a muattiaon in the g12d. a isomer of ras is HRAS or KRAS which accounts for more than fifty perceent of cancer. or we got g12c which makes gap not bind keeping ras on. 

so bascially scientist will study the ras/map tranduction pathway through the drosphila fly by looking at the ocmpound eye made up of ommitridum, it has 22 cell which has 8 neurons phohotsenosry. r1-r8 and the r8 is in the middle and the r1-7 are aorund it. r8 develops first and the r8 will serve as a system to differentiate the rest. so r8 will have proteins on the membrane that serve as ligands fro the other ones to differentiate. so r7 has a mutation which will lead to the inability to see uv light. so basically r8 has a moelcule on the membrane that is a ligan for the r7 rtk receptor called BOSS bride off sevenless. so bascallicy it will cause the receptor to homidimerze and then phoshphate itself and then have a grb2 bind called drk. drk has a sh2 domain also called 2sh3 and recognize the prhohylated tyronize and that recruits a (sos) called ras gef. so then that starts the ras singal tranduction. this helps the differentation of r7. 

a proto-oncogene is a gene that is normal and oncogene means that is hyperactivated. so onocgene is a prot ocnogene which potenitally can becme ono through a mutation that changes the cell fate to become tormignate. tumor supressos is a protein that prvent a cell from being tumorigenic bc it prevents a cell from going to those, becoming a tumor by slowing down or stoping the cell when needed. without ras the embyol will die but in the has of hypractivation in the rwong place and wrong time it can make ras a oncogene. RAS/ the receptor, map, mek, raf, mutaitons make it onco and phoahotse will work as a tumor supressor

gap not being able to bind to gtpase will make it onco and phooatste will make sure it stops by dephohroyalting the other pathways downstream. like RAF, MEK and MAP. cuz gap is not a kianse

son of sevenless is the SOS in the rastranduction

(ras gef)

so end of 3 

ALL OF FOUR 

HER is a 1-4 tryokinase kinase rkt transmembrane family. and they can lead to cancer due ot over hyperactivity of the her2 receptor. the anitbody to bind to the receptor to stop this is called herceptin. 

so studyig the mutated ras, g12d , or RASD scientist noticed that without ligan presenet/singal then cell proliferation still happebs. even if the cell is off and not active then proliferation doesnt happen. this indiciates that ras is downstream. anything upstream of ras doesnt matter. 

so they studied this through the observation of flies - they looked at the r8 /r7  . they took a fly double mutatnt. loss of fucnction for the receptor and hypeaactivation of the ras. this makes. adeveloped r7. loss of fucntion ras and hyperactive mek will lead to a developed r7. it is what is downstream what matters. upstreamd doenst matter as long as the hyperactivation kinase will go all the way through the mapk

GPCR is a recpeotr that is seven subintis. the c terminal end is in the cytosolic side and the n terminal end is in the exoplamic . what happens is that thsi protein is a mutlipass, and doesnt have enzymatic activity liek the rtk kinase. it is a cell surface and sometimes it can have a lipid anchor like a palmitic anchor. 

so a recpetor gpcr will have a ligan binded to it and then it changes confirmation shape and will be activaet for a downstream moclule. the seocn emmebran is a g protein compelx called 

G potien (gtpase protein complexf).it is a trimeric g rprotien complex. so when the grpcr activates what happens . So basically what happens is that receptor binds ligand, the gpcr and then ic change confirmation shape and allows for ABG the g protein complex to bind to it which is off. It binding to it the protein acts liek a GEF which will remove a gdp form the alpha and cause it to allow for a gtp to attach and release from the receptor itself and the BG secontion together. the hyperacivation of the gpcr  isnt going to cause cancer but it just cuases asthma or heart problems. its a fast resposne like immunie, smell, taste, seeing. 

g alpha is similar to ras. the gpcr is similar to gef. g alpha is similar to ras because it can activate the singal tranduction (sometimes is the beta gamma) . after g alpha is active gtp confirmaiton it can bind to an effector (molecule). so this effector can act as GAP (that speeds up the ras) and it can recyle g alpha because it can remove the gtp into gdp and then it can bidn back to the beta gamma and then start over ready for the next signal . 

so an effector is called adneyl cylase which is a transmembrane surface receptor that when bound to g alpha will make millions of secondary messanger camp and that will activate thosuands of protein kianse. and we need secondar messagers to amplify the singal and acitvate thousdands of prteoin kinase isntead of just one from a regular primary messanger. c amp are not protein material and have a short half life. so basically again it will go from gpcr then to the alpha g from the tripmeric g complex and then it will bind to the adenyl cylase - that will make cylic amp and then that will go and bind at  the r and prevent the c subuit of the protein kinase frombingin to the r and this will activate the kinase a. 

conetxt - 

so when we run or whatever we need need glycogen to become glucose to nrelease energy. so epinpherine will bind to the gpcr and then that will activate kinase and that will phosphorylate things that will breakdown glycogen to glucose. but not only does it phrohoylate the moclules that make glycogen to glucose but as well as prevents the revesre reaction of making glucose into glycogen. again by phrohylation . 

there are two dif types of a alpha there is g alpha S that is the excitator and then there is g alpha iwhich is an inhibtior. so when the body has enough energy and too much glucose what happens is that a ligand will bind to the gpcr and then it will make the g alpha i active and then that will bind to the same active adenyl cylase and then stop the reaction of making glycoten to glucose, 

So cholera will bin ot the g alpha s which will keep it on and cause diehra

Perutsisie wil bind to the g alpha i which will keep it off and make mucus

so if we just depend on the ligand degrading from the receptor it is bad because the ligand can have a long half life and will take a while. the receptors will get destinzed. a way to turn this off si through gpcr kianse which is different than the kianse that was activated that will bind to the receptor and then prhoylate it and cause a confrimation change and then it will lead to low affinity for the g alpha and then cause it to not bind and stop downstream. this gpcr kianse is called GRK

the gpcr kinase is called grk and will phohorylate the recpeotr and then it will cause b arrestin to bind along with other factors in order to induce endocytosis to engulf proteins on the memrbane along with those iwth ligands and then it iwll either recyle the receptors or it will kill them with lysosomes, and this is a negative system ffedback . 

so about nfkb it is a trancription factor that is used for immune repsone. it will activate genes . so cancer cells are addicted to nfkb because they need a lot to turn on genes that are going to keep the cancer cell alive. so the cell keeps the tf readily avialble in the cytoplasm waiting for a cell and once a bacteiral or viral singal comes in then the tf can go into the neuclues to turn on gene to fight against it. so the tf is rbough into the nucleus by an important protein complex and the inhibor is what holds it int hte cytoplasm and stops it from binding to the important yet. so in order to get rid of the inhibrot then the inrhibot is post tranlationally modified through ubiquination and gets degraded so the important complex can bind to the tf. 

so the NFKb is made up two proteins it is a heterodimer it si p50 and p65. in which the inhibrot called IKB i kappa b will bind and inhbit the transcription factor. 

so bascially a recpetor binds ligand and then it will cause the activation of a kinase called IKK. IKK will prhoylate the ikba inhibotro which has two prhoylation sites. the e3 will then reconzie (ligase) the site and then bring in a ubiquinator and then form a poly ubiquiatnor chain and then it can be recongized by a machinry to degrade. the e3 will add to the chain throguh covalent linkages. 

so to stop it , the nfkb needs to stop but activated it will make hundreds of poducts by activating geens and one of thsoe is a gene for ikba inhirbot which over time build up and then inhibit the nfkb. this is a repression model. 

so bascially the recptor and ligand bind and this causes the recptor to go through a confirmation change in whic traf5 can recognize and after it does then it leads to it self polyubqioantor because it is a 3e ligase. after it does that , the tail will serve as a platfrom for tac1 and ikk so tac1 can phsopaoylate ikk can turn it on. then ikk can go and prhoylat ikba and then e3 goes in and degrade . 

tac 1 binding doesnt depend on ikk and vise vesra. 

so the way that a poly ubqionation chain binds to the protein is througha. convalent bond and it is a glycine and a lysine. the glycine will be on the ubqiataont and hte lysine will be on the rpotein. if there are two chains it will be chyain on chain and it will still be glycine and lysine. the last glcycien of the second meolcule

so baswically to poly ubqinate there are three proteins. e1 through e3. e1 is the activator that uses atp and then it will take it and pass it to e2 which will bind o the protein and these two are not speicif. then e3 which is pseicif to the protein will bind and tranfer the ubiq from the e2 to the protein and bind glycien to lysien in the protein. and thsi iwll confute. the protein needs to have five or four in order to be degradeable. 

so these sequences are the dbox(destruction) , ken box, pest box, and hdyrphobic path. after the inhibrot gets phroylated it will expose a sequence in which the e3 will recognize. ah so 

the 26s proteomosn is a complex to do degradation. it is made out of the 20s core and the 19s caps. so the 20s core is made up of four rings , two alpha and two beta wich are in the middle. each ring has 7 subunits. the beta ones are asymetrical and they have subtuni 1,2 as to cleave acidic and basic residues, and the 5th one is hydrophobic residues. then there is the alpha surrounding them. the inside of the 20s barrel is hollow and the middle has the catyalic actity. it is 5nm in dimaeter/tall because it needs to be small to make the poly u chain go into as linear so it is easeir to degrade. the aloha rinds to not have catlyci actiivty. 

so the cap is called 19s or pa700 which has three reagions. the first domain is where the protein will reocnize the polyUBiq fromanother protein andbind, two will use DUBS in order to take the chain from the protein, and three will use atp to unwind the protein and put it in the chamber. 

Different linkage form in a change that can be reconzed as different singal that indicate degradation. certain cap will reconzie the 48 linkage change for degration. remeber that lysien 48 is primarly the target which is used for dgeation. so lysein 11 is for something else. not for degradation tho. 

lecture 5 -

so the tranportation of moclules happens through nucelopores - NPCS and they are in the nuceloenvelope of the nucleous. the evelope is made of a bilayer that has ONM on the cytoolis and the INM in the nuclous side. NPC’s are made out of nuceloproiens which are a group of 30 proteins but each one has 12-36 copies. 

these nuceloporines are made up of amino acids which special fucntios either for the filaments or for the basekts. the filaments are faking the cytosolic side and the basket is in the neuclous. some of them are for the tranport of mcoluel and other are for the strucutre. 

there are fg nuceleoporins that are hydropohbic and are located in the middle channel of the NPC. they have hydrophobic amino acids. and looks like sphagetti all jumbled up. Every cell type has different number of NPC and types and iwthout NPCs then the cell cannot survivie. therefore cancer cells have a lot more to tranport effienclty. 

so the NPC tranports in and out at the same time all at once and fast. it moves in 10^6 power moclule and a bunch. it is also not specific for the protein type. but it does regulate the size of the mcoleul that can diffuse in or out. so if its 40dka and smallere it can diffuse on its own if its bigger it will be slower and need help. 

a big multicomplex protein will need help to pass and it will go through slow as a whole so it donest go and disassmebl and ressemble. no there is no need for that. 

so bascailly what scinetist did is that they wanted to see how the molcuels flow into the and out of the nucelous. they took nuceloplasmin and xenopus ooyctes. they took nthe ncueloplasmin complex and amplified it and made it big and isolated it. these are made of a ncueoplamsi which is five heads and five tails. then they used protease to cut them and put them in a gel to sperate the size. they took the sizes which is tail, head, head and tail, and all five of them that make up a complex and injected int into the cytosplams. they took the head/core and put it in the cytoplasm with stains and saw no mocment, then they took that head and put it in the ncuelous just to see if it like degraded realy fast but it was still there. then they took the tails and put that in the cytoplams and saaw movment, then they did head and tailn and saw movemnt. this revleaed that the tail has a amino acid sequence called LNS.  the tail itself is too small so it will difuse in either way so that is why did they did head and tial. 

they discoverd this NLS sequnece through the SC40 virus which has a 40tlarge antigen that will allow the oncogenic gene go int the ncuelus of the host cell and when mutated it will no logner go. the nls sequence has seven amino acids. althoguh there are different types of sequences. 

Scientist took thus kinase pruvate protein that is loalized in the cytoplasm and they added thsi sc40tanitgen which has the NLS and then they saw that it went into the nucelus showing that mcoluel with NLS will go itno the ncuelus. 

so digitonin was used to make holes in the cytoplasm and then what they did is add flouscents to the NLS. the cytoplasmid proteins all left and the nls didnt go inside the ncuelus. but if the factors are inside the ncuelus the nls does go inside the nucleus showing that the NLS on its own isnt suffient enough to import. 

NES is what export cytoplasm to nucleus. nls these cant go inside by themlves and need helper proteins by piggy back riding.