Cleaved parp c parp

After various treatments as indicated, cancer cells were harvested, and the total protein was lysed in radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) and then quantified by a BCA kit (Epizyme, Shanghai, China). Equal amounts of proteins were resolved on 10%-12.5% SDS-PAGE gels, followed by transferring the proteins to an Immobilon PVDF Membrane (Merck Millipore Ltd, Tullagreen, Ireland). PVDF membranes with proteins were blocked with 5% skim milk for 1 hour, incubated with primary antibodies overnight at 4°C, and then conjugated with secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 hour. Signals were visualized by ECL reagent (Epizyme, Shanghai, China) and photographed by Tanon 5200 visualizer (Tanon, Shanghai, China).
The primary antibodies used are as follows: p27, p21, Cyclin D1, PARP, cleaved PARP (c-PARP), cleaved caspase 3, cleaved caspase 9, cleaved caspase 7, cleaved caspase 8, Noxa, Bak, Bax, Bik, Bim, Bad, Puma, Bcl2, Bcl-xl, Mcl-1, XIAP, BID, TRAIL, DR4, DR5, CHOP, ATF4, eIF2α, and p-eIF2α were from Cell Signaling Technology (USA). Cyclin E, CDK2, CDK4, CDK6, Fas, DR3, c-Myc, and p53 were from Santa Cruz Biotechnology (USA). TNFR1 and TNFR2 were from Proteintech (USA). β-Actin was from HuaBio (China).

Cisplatin (DDP, Cat.^#H20010743) injection was purchased from Jiangsu Hausen Pharmaceutical Co., Ltd (Jiangsu, China). Dulbecco's modified Eagle's medium (DMEM, Cat.^#11965092), fetal bovine serum (FBS, Cat.^#10270106), penicillin/streptomycin (Cat.^#10378016), and phosphate-buffered saline (PBS, Cat.^#10010023) were supplied by Gibco (NY, USA). Primary antibodies against Beclin1 (Cat.^#11306-1-AP), p62 (Cat.^#66184-1-Ig), Cytochrome c (cyto. C, Cat.^#66264-1-Ig), and DAPK1 (Cat.^#25136-1-AP) were purchased from Proteintech (Wuhan, China). Primary antibodies against Phospho-AMPKα (p-AMPKα, Cat.^#50081S), Phospho-mTOR (p-mTOR, Cat.^#5536T), Atg7 (Cat.^#8558T), LC3A/B (Cat.^#12741S), Cleaved Caspase-9 (c-Casp 9, Cat.^#20750S), Cleaved Caspase-3 (c-Casp 3, Cat.^#9661T), Cleaved PARP (c-PARP, Cat.^#5625T), Phospho-SAPK/JNK (p-JNK, Cat.^#9255S), Phospho-p53 (p-p53, Cat.^#9284T), Bax (Cat.^#2772T), and GAPDH (Cat.^#5174T) and rabbit (Cat.^#7074P2) or mouse (Cat.^#7076P2) secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Cells were harvested and lysed in ice-cold RIPA lysis buffer with added protease and phosphatase inhibitor (Cell Signaling Technology, Danvers, MA USA). Equal amounts of protein were resolved and separated on an SDS/PAGE gel (BioRad TrisGlycine 4–20% gel), transferred onto PVDF membranes, and subjected to immunoblot analyses. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline, pH 7.4, containing 0.05% Tween 20, and were incubated with primary and secondary antibodies according to the manufacturer’s instructions. Blotting was performed using antibodies targeting PLK1 (Abcam, Cambridge, UK; Cat. #: 17056), DNA methyltransferase 1 (DNMT1) (BD Biosciences, San Jose, CA USA; Cat. #: 612618), Bcl-xL (Cell Signaling; Cat. #: 2764), Cleaved PARP (c-PARP) (Cell Signaling; Cat. #: 9541), p53 (DO-1) (Santa Cruz, Cat. #126), p-p53 (Ser15) (Cell Signaling; Cat. #: 9284), STAT3 (BD Biosci, Cat. #: 610189), p-STAT3 (Tyr705) (abcam; Cat. #: 76315), Cleaved Caspase-7 (c-Caspase-7) (Cell Signaling; Cat. #: 9491), Cleaved Caspase-3 (c-Caspase-3) (Cell Signaling; Cat. #: 9541), and Vinculin (Cell Signaling; Cat. # 4650). Dilutions used for Cleaved Caspase-3 and Cleaved Caspase-7 were 1:250, and for other antibodies were according to the company’s recommendations.

Primary antibodies to the following proteins were used: cleaved PARP (c-PARP), cleaved caspase 8 (c-caspase 8), cleaved caspase 9 (c-caspase 9), ATF4, CHOP, Noxa, DR3, Bax, Bad, Bid, Bim, p53, p21, p-histone 3 (p-H3), p-H2AX, p-cdc2 (Cell Signaling Technology, Danvers, MA, United States); DR5 and FoxO3a (Abcam, Cambridge, MA, United States); TNFR1, TNFR2 and β-actin (HuaBio, China).

Antibodies against AKT, phosphorylated AKT at S473, ERK1/2, phosphorylated ERK1/2 (T202/Y204), mTOR, phosphorylated mTOR (S2481), p70S6K, phosphorylated p70S6K (T421/S424), cleaved caspase-3 (C-Cas3), cleaved caspase-9 (C-Cas9), and cleaved PARP (C-PARP) were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against GAPDH and Tubulin were obtained from Huaan Biotechnology Co., Ltd. (Hangzhou, China). Z-VAD-fmk was obtained from Selleck Chemicals (Houston, TX, USA). Muse™ Cell Cycle Kit and Muse^® Annexin V & Dead Cell Kit were purchased from Millipore (Billerica, MA, USA). Other reagents were purchased from Beyotime Biotechnology, Shanghai, China.