LRRK2 Kinase Inhibitor Rejuvenates Oxidative Stress-Induced Cellular Senescence hauv Neuronal Cells Part 1

Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv

Keeb kwm.Leucine-nplua nuj rov kinase 2 (LRRK2) ua lub luag haujlwm tseem ceeb hauv kev tsim cov kab mob Parkinson's disease (PD). Kev laus yog qhov tseem ceeb tshaj plaws rau kev loj hlob ntawm PD. Kev sib raug zoo ntawm kev laus thiab cellular senescence tau tsim. Cellular senescence yog correlated nrog dysregulation ntawm txoj kev proteolytic thiab mitochondrial dysfunction, uas kuj yog txuam nrog aggregation ntawm -synuclein (-syn). Tib neeg dopaminergic neuron-zoo li hlwb (sib txawv SH-SY5Y hlwb) tau kho nrog rotenone nyob rau hauv lub xub ntiag los yog tsis muaj LRRK2 kinase inhibitor GSK2578215A (GSK-KI) rau 48 h. Cov cim ntawm cellular senescence, suav nrog p53, p21Wafi/Cip1(p21), -galactosidase(-gal), Rb phosphorylation, senescence-associated (SA) -gal kev ua, thiablysosomal uakev ua, raug tshuaj xyuas. Cov hlwb khiav ceev thiab cov nas thawj cortical neurons tau kho nrog -syn fibrils 30 min ua ntej kev kho mob nrog rotenone nyob rau hauv lub xub ntiag los yog tsis muaj GSK-KI rau 48 teev. Cov nas tau txhaj tshuaj intraperitoneally nrog rotenone thiab MLi-2 (LRRK2 kinase inhibitor) ib zaug txhua ob hnub rau ob lub lis piam. Cov txiaj ntsig. Rotenone upregulated LRRK2 phosphorylation thiab -gal theem los ntawm kev ua kom lub p53-p21 signaling axis thiab downregulated Rb phosphorylation. Tsis tas li ntawd, rotenone upregulated SA -gal kev ua, reactive oxygen hom qib, thiab LRRK2phosphorylationthiab inhibited lysosome kev ua haujlwm. Rotenone-induced LRRK2 upregulation impaired lub clearance ntawm a-syn fibrils. Kev kho mob nrog LRRK2 inhibitor mitigated rotenone-induced cellular senescence thiab -syn tsub zuj zuj. Cov lus xaus. Rotenone-induced upregulation ntawm LRRK2 kinase kev ua haujlwm txhawb nqa cellular senescence, uas txhim kho -syn tsub zuj zuj. Txawm li cas los xij, kev tswj hwm ntawm LRRK2 kinase inhibitor rejuvenated rotenone-induced cellular senescence.

Thov nias ntawm no kom paub ntxiv

1. Taw qhia

Parkinson's disease (PD), uas yog cov kab mob neurodegenerative feem ntau, yog tus cwj pwm los ntawm kev tswj lub cev tsis muaj zog[1]. Ntau yam caj ces thiab ib puag ncig ua rau muaj qhov tshwm sim ntawm PD[2-5].LRRK2, qhov tseem ceeb ntawm kev pheej hmoo rau PD, nthuav tawm GTPase thiab kinase cov dej num [6]. LRRK2 G2019S mutant, uas nthuav tawm cov kev ua kinase txhim kho, txhawb nqa kev nce qib PD [7]. Qhov kev pheej hmoo ntawm kev tsim PD hauv cov neeg mob harboring G2019S mutant nce nrog hnub nyoog [8] raws li kev laus yog txuam nrog kev loj hlob ntawm cov kab mob neurodegenerative [9]. Yav dhau los, peb tau tshaj tawm tias LRRK2 kinase kev ua haujlwm txhawb nqa cellular senescence thiab inhibited degradation ntawm alpha-synuclein (-syn) aggregates [10]. -Syn yog ib feem tseem ceeb ntawm Lewy lub cev (LB) lossis Lewy neurites, uas yog cov cim tom qab ntawm PD. Impaired degradation ntawm -syn ua rau nws aggregation [11].

Cellular senescence impairs protein degradation machinery[12-14]. Kev raug rau cov tshuaj rotenone tsawg, uas tau tshaj tawm los ua rau PD, txhawb nqa cellular senescence nyob rau hauv tib neeg trabecular meshwork cell kab [15]. Tsis tas li ntawd, rotenone upregulates LRRK2 kinase kev ua haujlwm hauv cov neurons [16]. Yog li, peb xav tias rotenone txhawb nqa cellular senescence los ntawm kev ua kom LRRK2 kinase thiab thiaj li txhim kho -syn aggregation.

Cistanche tuaj yeem txhim kho kev tiv thaiv

2. Cov txheej txheem thiab cov khoom siv

2.1. Cell Kab lis kev cai thiab kev kho mob.

Tib neeg neuroblastoma cell kab (SH-SY5Y hlwb) tau coj mus rau hauv ib qho kev loj hlob nruab nrab (Dulbecco's modified Eagle's nruab nrab (DMEM;10-013-CV; Corning cellgro, Thermo Fisher Scientific, Waltham, MA, USA) ntxiv nrog 1{{ 48}} feem pua ​​​​fetal bovine serum (FBS, 35-010-CV, Corning cellgro), 1 feem pua ​​penicillin/streptomycin(P/S, 10378-016, Gibco, Thermo Fisher Scientific), thiab 1 feem pua ​​mycoplasma tshem tawm reagent (KOMA)) ntawm 37 degree hauv 5 feem pua ​​CO, incubator (Thermo Fisher Scientific). Qhov sib txawv SH-SY5Y hlwb (dSH hlwb) tau txais tom qab kev kho mob nrog 10μM all-trans retinoic acid (R2625-100MG, Sigma-Aldrich, St. Louis, MO, USA) npaj nyob rau hauv ib qho kev loj hlob nruab nrab ib zaug txhua ob. hnub rau xya hnub. Hnub 7 tom qab kho retinoid acid, cov hlwb raug kho nrog rotenone (1 uM) thiab GSK2572815A (GSK-KI; LRRK2 kinase inhibitor; 1μM) pre-pared hauv qhov nruab nrab loj hlob rau 48 teev. Dimethyl sulfoxide (DMSO) tau siv los ua lub tsheb rau rotenone thiab GSK-KI. Cov-syn fibrils tau huv si raws li tau piav qhia yav dhau los [10]. Cov rotenone- thiab GSK-KI-kho cov hlwb tau kho nrog a-syn fibrils (70nM). Cov nas thawj cortical neurons tau kho nrog rotenone (1μM), GSK-KI (l μM), thiab a-syn fibril (70nM) rau 48h. Cov kev kho mob rau cov nas thawj cortical neurons tau zoo ib yam rau cov neeg ua haujlwm rau dSH. Cov hlwb raug ntxuav ob zaug nrog dej khov-txias phosphate-buffered saline (PBS) thiab lysed nyob rau hauv 1x qauv buffer (50mM Tris-HCl (pH6.8), 2% sodium dodecyl sulfate, 10 feem pua ​​glycerol, 1 feem pua ​​-mercaptoethanol, thiab 0.02 feem pua ​​​​bromophenol xiav).

2.2.Kev cais thiab kab lis kev cai ntawm Primary Cortical Neurons los ntawm E16 Rat Embryos.

Cov nas cev xeeb tub tau euthanized nrog CO, roj. Lub tsev menyuam tau raug txiav tawm, thiab cov fetuses los ntawm lub hnab embryonic tau muab tso rau hauv cov dej khov txias HBSS-/-(14175-079, Gibco, Thermo Fisher Scientific). Lub pob txha taub hau tau tev tawm siv forceps, thiab cov cortices tau txiav tawm tag nrho lub hlwb. Tom qab tshem tawm cov meninges, cov cortices tau incubated nrog 0.25 feem pua ​​Trypsin-EDTA(25200-056, Gibco, Thermo Fisher Scientific) ntawm 37 degree C rau 20min hauv ib da dej. Tom ntej no, cov cortices tau incubated nrog 40ug / mL DNase I (DN25, Sigma-Aldrich), vortexed maj mam, thiab incubated rau 5min nyob rau hauv ib tug 37 degree da dej. Feem ntau ntawm cov supernatant raug tshem tawm los ntawm kev nqus, thiab cov qauv tau tsim nrog cov tshuaj tiv thaiv kab mob uas muaj MEM (11090-08, Gibco, Thermo Fisher Scientific), 1.5% DMEM, 5% FBS, 2.5mg / mL bovine serum albumin. (BSA; A7906, Sigma-Aldrich), thiab 2.5 mg/mL trypsin inhibitor (T9253, Sigma-Aldrich). Cov tshuaj tua kab mob tau raug tshem tawm, thiab cov cell pellet tau rov ua dua hauv 10 npaug ntawm nws qhov ntim nrog qhov nruab nrab loj hlob. Cov muaj pes tsawg leeg ntawm kev loj hlob nruab nrab yog raws li nram no: neurobasal nruab nrab (21103-049, Corning cellgro, Thermo Fisher Scientific), 2 feem pua ​​B-27(17504044, Corning cellgro, Thermo Fisher Scientific), thiab 1x GlutaMAX{ {34}}(35050-061, Corning cellgro, Thermo Fisher Scientific). Cov nas uas raug rho tawm thawj cortical neuron hlwb (3 × 10 degree /well) tau cog rau hauv 12-zoo phaj (SPL30012, SPL Life Sciences, Pochoen, Kaus Lim Qab Teb) thiab coj mus rau qhov nruab nrab loj hlob ntawm 37 degree hauv 5 feem pua ​​CO, incuba-tor rau 24h. Cov kab lis kev cai nruab nrab tau hloov nrog kev loj hlob nruab nrab ntxiv nrog 0.2mg / mL ntawm 5-fluoro-2'-deoxyuridine (F0503, Sigma-Aldrich) thiab 96ug / mL ntawm uridine (U3750, Sigma-Aldrich) rau inhibit mitosis. Hnub 6, cov hlwb raug kho nrog 1uM rotenone, 1uM GSK-KI, thiab 70nM -syn fibrils rau 48h thiab sau nrog lx qauv tsis.

2.3. Western Blot.

Western blotting tau ua raws li tau piav qhia yav dhau los [17]. Cov tshuaj tiv thaiv hauv qab no tau siv rau sab hnub poob blotting: luav anti-LRRK2 phospho-S935 monoclonal (ab133450, Abcam, Cambridge, UK), luav-ntsis anti-LRRK2 phospho-S1292 monoclonal (ab203181, Abcam), nas monoclonal (2) N241A/34, Neu-roMab, UC Davis, CA, USA), nas anti-p53 monoclonal (rau tib neeg p53: sc-126; rau nas p53: sc-393031; Santa Cruz Biotechnology, Dallas, TX, USA), nas anti-p21wafi/-Ccip1(p21) monoclonal(CM5131, ECM Biosciences, Ver-sailles, KY, USA), nas anti-Rb monoclonal(#9309S, Cell Signaling Technology(CST), Danvers, MA , Tebchaws USA), anti-phosphoRb(Ser807/811)(#9308S, CST), nas anti- -galactosidase monoclonal (sc-377257,Santa Cruz Biotech-nology), nas anti-p62 monoclonal(ab56416) , Abcam), nas anti- -actin monoclonal(sc-47778; Santa Cruz Bio-technology), luav anti-LC3B polyclonal(#2775S; CST), nas anti- -syn (clone 42) Monoclonal (610786; BD Bio-sciences, San Jose, CA, USA),Anti-phospho-threonine-argi-nine(#2351S, CST), anti-p53(SC-99, Santa Cruz Biotechnology), anti-LaminB(SC-6216}, Santa Cruz Biotech-nology), tshis peroxidase-conjugated AffiniPure anti-nas IgG(H plus L)(#115-035-003; Jackson Immunoresearch Labo-ratories Inc., West Grove, PA, USA), thiab tshisperoxidase-conjugatedAffiniPure anti-Lavbit IgG(H plus L)(#1l1-035-144; Jackson Immunoresearch Laboratories Inc.) cov tshuaj tiv thaiv. Immunoreactive signals nyob rau hauv nitrocellulose membrane tau tsim nrog Luminata Crescendo Western HRP (#WBLUR0500, Merck & Co. Inc., Kenilworth, NJ, USA), thiab cov dluab raug ntes nrog lub koob yees duab MicroChemi4.2 (Shimadzu, Kyoto, Nyiv).

2.4.Proximity Ligation Assay (PLA) thiab Immunofluorescence (IF).

PLA tau ua tiav siv Duolink⑨ Hauv Situ Detection Reagents Green (DUO{{0}}}RXN, Sigma-Aldrich), DuolinkeIn hauv Situ PLA4Probe Anti-Mouse MINUS antibody (DUO{92002-100}RXN, Sigma-Aldrich) , thiab Anti-Rabbit Plus antibody (DUO92004-100RXN, Sigma-Aldrich), ua raws li cov chaw tsim khoom cov lus qhia. Cov hlwb SH-SY5Y tau cog rau hauv 96- cov phiaj hlau zoo dub (655077, Greiner Bio-One, Krems-münster, Austria) tau sib txawv, thiab cov hlwb sib txawv tau kho nrog rotenone thiab GSK-KI hnub 7. Cov hlwb tau muab yaug ob zaug nrog dej khov-txias Dulbecco's PBS (DPBS), kho nrog 4 feem pua ​​​​ntawm paraformaldehyde rau 30 min ntawm chav tsev kub (RT), thiab yaug peb zaug nrog dej khov-txias DPBS. Tom ntej no, cov hlwb tau permeabilized siv 0.1 feem pua ​​Triton X-100 npaj hauv DPBS. Tom qab ntxuav peb zaug nrog txias DPBS, lub hlwb tau incubated nrog ib tug poob ntawm thaiv cov tshuaj ntawm 37C rau 1h. Cov tshuaj thaiv thaiv tau raug tshem tawm, thiab cov hlwb raug tsim nrog cov tshuaj tiv thaiv (50μL) ntawm 37 degree rau 3h. Cov tshuaj tiv thaiv lub cev muaj xws li cov tshuaj tiv thaiv kab mob diluent los ntawm cov khoom siv (cov tshuaj tiv thaiv siv rau PLA: nas anti-LRRK2 mono-clonal (1:20) antibody thiab luav anti-LRRK2 phospho-S1292 monoclonal (1:20) antibody; antibody siv rau IF assay: qaib anti- -gal polyclonal antibody (1:400, ab9361, Abcam). Tom qab ntawd cov qauv raug ntxuav ob zaug nrog 1x ntxuav tsis tau A rau 5min.Rau npaj cov tshuaj sojntsuam, Duolink⑧In Situ PLA · Kev Tshawb Fawb Anti-Mouse Minus thiab Anti-Lavbit Plus tau tov nrog cov tshuaj tiv thaiv kab mob diluent (1:5). Rau qhov IF assay, cov tshuaj tiv thaiv - -gal antibody kuj tau ntxiv (1:400) rau qhov sib tov. Cov hlwb raug incubated nrog 50μL ntawm cov tshuaj sojntsuam ntawm 37C rau 1h. Tom qab ntawd, cov hlwb raug ntxuav ob zaug nrog lx ntxuav tsis pub A rau 5 feeb. Rau qhov IF assay, ligase (40: 1) thiab anti- -gal antibody (400: 1) tau sib xyaw rau hauv ligation tsis. Cov hlwb raug incubated nrog ligation tov (50μL) ntawm 37 degree rau 30min thiab ntxuav ob zaug nrog lx ntxuav buffer A rau 5min. Rau IF assay,polymerase(1:40) tau incubated nrog tshis Anti-Chicken IgY H plus L(Alexa Fluor② 594; ab150172) Secondary antibody (1:500) rau 100min ntawm 37C. Cov hlwb raug ntxuav ob zaug nrog 1x ntxuav tsis B rau 10 feeb, tom qab ntawd ntxuav nrog 0.01x ntxuav tsis B rau 1 feeb. Txhawm rau stain lub nucleus, cov hlwb tau tsim nrog Hoechst 33342 (1: 1000; 62249, Thermo Fisher Scientific) npaj hauv DPBS ntawm RT rau 10min. Cov duab ntawm cov hlwb hauv 96- cov phiaj hlau zoo tau raug ntes tau siv lub tshuab ntsuas hluav taws xob confocal laser (LSM 700, Carl Zeiss Jena, Lub teb chaws Yelemees).

2.5.Immunoprecipitation.

Tag nrho cov cell lysates ntawm dSHcells kho nrog rotenone (1 μM) thiab GSK-KI (1 uM) tau ua ntej siv cov tshuaj lysis (PBS, 1 feem pua ​​Triton X-100, 1x protease inhibitor cocktail, thiab lx phosphatase inhibitor cocktail). Cov lysates tau centrifuged ntawm 4, 000g thiab 4 degree degree rau 5min, thiab lub supernatant tau incubated nrog anti-p53 antibody (554293, BD Biosciences) thiab PierceProtein G Aga-rose (20398, Thermo Fisher Scientific) resuspended hauv cov tshuaj lysis rau 12h. Cov hlaws tau raug ntxuav ob zaug nrog cov tshuaj lysis, thiab cov hlaws-protein complex yog denatured nrog lx qauv tsis.

2.6. Nuclear Fractionation.

Cov feem nuclear raug cais tawm siv NE-PERr Nuclear thiab Cytoplasmic Extraction Reagents (78833, Thermo Fisher Scientific), ua raws li cov chaw tsim khoom cov lus qhia.

2.7. mRNA Kev Npaj thiab Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).

mRNA raug cais tawm ntawm rotenone / GSK-KI-kho dSH hlwb siv cov khoom siv RNeasy Plus Mini (74134, QIAGEN, Hilden, Lub teb chaws Yelemees). Tom ntej no, mRNA tau thim rov qab rau hauv cov DNA sib ntxiv (cDNA) siv TOPscript cDNA Synthesis Kit (EZ005S, Enzynomics, Daejeon, Republic of Kauslim).cDNA raug rau qRT-PCR siv TOPreal"qPCR 2x Pre-MIX (SYBR Green nrog qis. ROX, UDG plus)(RT500M, Enzynomics). Cov primers hauv qab no tau siv rau qRT-PCR: human p21,5'-ATG AAA TTC ACC CCC TTT CC-3'(forward)) thiab 5'-CCC TAG GCT GTG CTC ACT TC-3'(reverse); human p16INK4(p16),5'-CCC AAC GCA CCG AAT AGT TAC-3'(forward) and 5'-CAC GGG TCG GGT GAG AGT -3'(rov qab).qRT-PCR tau ua nyob rau ntawm Mic qPCR cycler(MIC-4, BioMolecular Systems, Upper Coomera, Australia).

2.8. Kev ntsuas ntawm Senescence-Associated (SA) Galactosidase (-Gal) Kev Ua Haujlwm, Reactive Oxygen Species (ROS), thiab Lysosome Activity.

Thaum 30 feeb tom qab kev kho mob, cov hlwb nyob dSH tau stained nrog 2μM GlycoGREEN- Gal (GC611, Goryo Chemical Inc., Hokkaido, Nyiv), 5uM 2',7'-dichlorofluorescein diacetate (DCFDA; 287810, Calbio-chem, CA, San USA), 2μM LysoSensorm Blue DND-167 (L7533, Invitrogen, Carlsbad, CA, USA), thiab 1 μM Hoechst 33342 rau kev tshuaj xyuas SA-gal kev ua, ROS, nquag lysosomes, thiab nucleus, ntsig txog. mounted nrog ProLongDiamond Antifade Mountant (P36965, Invitrogen) thiab duab siv lub confocal laser scanning microscope. Cov duab sib txawv ntawm cov duab sib txawv tau txais los ntawm kev siv lub tshuab tsom teeb confocal laser scanning microscope. {21}}zoo cellular senescence assay kit(CBA-213, Cell Biolabs Inc., San Diego, CA, USA), ua raws li cov chaw tsim khoom cov lus qhia.

2.10.Mus tuav thiab tshuaj noj.

Cov nas tau ua raws li tau piav qhia yav dhau los [18] raws li cov lus qhia ntawm Dankook Animal Ethics Committee (Dankook IACUC,18-026).C57BL/6] cov nas txiv neej hnub nyoog 16 lub lis piam tau txhaj tshuaj intraperitoneally nrog rotenone (0 .75 mg/kg lub cev hnyav) thiab MLi-2 (ib qho LRRK2 kinase inhibitor; 1mg/kg lub cev hnyav) ib zaug ob hnub rau ob lub lis piam.

2.11.Rotarod Test thiab Brain Tissue Npaj.

Hnub 14 tom qab kev kho mob, cov nas tau muab tso rau ntawm lub tog raj kheej kheej kheej kheej kheej. Lub sijhawm yuav tsum tau poob rau hauv pem teb tau sau tseg. Cov ntsiab lus ntawm qhov kev xeem rotarod tau piav qhia lwm qhov [18]. Tom qab ntawd, cov nas tau euthanized thiab transcardially perfused nrog dej txias HBSS uas muaj Ca2 thiab Mg. Lub midbrain tau dissected nrog micro txiab thiab tweezers. Cov ntaub so ntswg tau lysed hauv PBS uas muaj 1 feem pua ​​Triton X-100,1xXpert Protease Inhibitor Cocktail Solution (P3100, GenDEPOT, Katy, TX, USA), thiab lx Xpert Phosphatase Inhibitor Cocktail Solution (P3200, GenDEPOT). Cov qauv no tau homogenized siv Pellet Pestle Cordless Tsav (Sigma-Aldrich). Tus supernatant tau raug rau sab hnub poob blotting, SA -gal kev ua ub no, thiab enzyme-linked immunosorbent assay (ELISA).

2.12.ELISA rau Oligomeric -Syn.

Yav dhau los, peb tau tsim ib lub qhaub cij ELISA rau kev tsom xam ntawm fibrillar -syn oligomers siv cov tshuaj tiv thaiv kab mob uas lees paub cov filamentous conformation ntawm -syn aggregates [17]. Cov crude midbrain lysates tau raug rau ELISA, thiab -syn aggregates tau suav nrog -syn fibril qauv.

2.13. Cov duab thiab cov ntaub ntawv tsom xam.

Qhov kev siv ntawm PLA thiab nyob-cell staining tau ntsuas siv Zen 2012 (Carl Zeiss). Densitometry ntawm lub hom phiaj proteins tau ua los ntawm kev siv

Daim duab 1: Rotenone-induced LRRK2 kinase activation txhawb nqa cellular senescence nyob rau hauv sib txawv tib neeg neuroblastoma cel kab. Western blotting tsom xam ntawm dSH hlwb kho nrog 1 uM rotenone los yog 1 uM GSK2578215A (GSK-K), ib qho LRRK2 kinase inhibitor, rau 48 h siv cov tshuaj tiv thaiv tiv thaiv lub hom phiaj proteins(a). Qhov ceev ntawm cov proteins tau normalized rau cov -actin (bg).n =3.(h) dSH hlwb kho nrog 1 uM rotenone lossis 1uM GSK-KI rau 48h tau raug rau qhov sib thooj ligation assay (PLA) ntawm phospho-S1292 LRRK2 thiab tag nrho IRRK2 (PIA-pS1292) thiab immunofluorescence (TF) tsom xam ntawm -galactosidase, Nuclei tau stained nrog Hoechst 33342.Cov kev tswj siv hauv PLA thiab IF staining qhia qhov siv tau ntawm cov txiaj ntsig (txoj cai ob lub panels). Qhov kev siv ntawm PLA-pS1292(i) thiab -galactosidase (j) tau normalized rau cov ntawm 4', 6-diamidino-2-phenylindole.n= 4; tus lej ntawm cov cell=12-17.

Multi Gauge (Fujilm, Tokyo, Nyiv, Nyiv).Txhua cov ntaub ntawv tau txheeb xyuas thiab teeb tsa siv Prism 8 (GraphPad Software, San Diego, CA, USA). Cov ntaub ntawv los ntawm kev sim ua siv dSH hlwb thiab nas thawj cortical neurons tau txheeb xyuas siv ob txoj kev tsom xam. ntawm qhov sib txawv (ANOVA), ua raws li Bonferroni qhov kev xeem hoc (n=3). Lub caij no,the data obtained from mouse experiments, live-cell stain-ing,and PLA in the dSH cells were analyzed using two-way ANOVA, followed by Tukey's post hoc test (n>3). Tag nrho cov ntaub ntawv yog sawv cev raws li txhais tau tias ± tus qauv yuam kev ntawm txhais tau tias. *p<><><><0.0001,and n.s.="not">

Daim duab 2: LRRK kinase inhibitor inhibits kev ua haujlwm ntawm p53 hauv cov kab sib txawv ntawm tib neeg neuroblastoma cell kab. (a) Tag nrho cov cell lysates (20 feem pua), uas tau siv los ua cov tswv yim ntawm immunoprecipitation (IP), thiab cov khoom siv hluav taws xob tau sau. Qhov kev tshuaj ntsuam tau qhia tias 20 feem pua ​​​​ntawm cov tswv yim tau nthuav tawm cov txiaj ntsig zoo ib yam li cov uas tau pom hauv daim duab 1. (b, c) Cov qauv IP raug denatured thiab raug rau sab hnub poob blotting. TXR site phosphorylation theem hauv p53 tau normalized rau tag nrho p53 qib. n=3.(de)Tag nrho p53 qib nyob rau hauv lub nuceus raug tshuaj xyuas siv western blotting. LaminB tau siv rau normalization ntawm nuclear p53 qib. n=3.(f) Cov qib mRNA ntawm p21 thiab pl6 hauv pawg kho mob tau ua rau cov neeg nyob hauv lub tsheb-(dimethyl sulfoxide-) kho pab pawg, n =3.

Kab lus no yog muab rho tawm los ntawm Hindawi Oxidative Medicine thiab Cellular Longevity Volume 2021, Tshooj ID 9969842, 16 nplooj ntawv https://doi.org/10.1155/2021/9969842