Pbcl 2 s70

Cell lysates were extracted with cell lysis buffer (Beyotime Biotechnology, Nantong, China), and the protein concentration in the lysates was quantified using a Micro BCA Protein Assay Kit (Sangon Biotech, Shanghai, China). A total of 20–50 μg of each cell lysate sample was loaded for immunoblotting and detected by antibodies that recognize Tubulin, VDAC, AIF, SMAC/DIABLO, cytochrome c (Epitomics, Hangzhou, China), CUL1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CDKN1B/P27, CDKN2A/P16, PARP, γH2AX, caspase3, caspase8, caspase9, BCL-2, pBCL-2 (S70), BCL-XL, MCL-1, BID, BAD, BAX, and BIM (Cell Signaling Inc., Danvers, MA, USA).

Quantitative PCR (qPCR) and western blotting were performed as described previously.21 FSTL5 primers: forward 5′‐AACAACTCACGCTTCAAG‐3′; reverse 5′‐TGTATGCTCCAGTATCTTCA‐3′. β‐actin primers: forward 5′‐TGGACTTCGAGCAAGAGATG‐3′; reverse 5′‐GAAGGAAGGCTGGAAGAGTG‐3′. The following primary antibodies were used in the western blotting assays:Anti‐FSTL5 antibody (1:1000; Sigma, St. Louis, MO, USA), β‐actin (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), proliferating cell nuclear antigen (PCNA, 1:1000; Santa Cruz Biotechnology), Cleaved Caspase‐3 (1:1000; Cell Signaling, Boston, MA, USA), Cleaved Caspase‐8 (1:1000; Cell Signaling), Cleaved Caspase‐9 (1:1000; Cell Signaling), Bax (1:1000; Cell Signaling), Bad (1:1000; Cell Signaling), Puma (1:1000; Cell Signaling), P‐Bcl2(S70) (1:1000; Cell Signaling), P‐Bcl2(T56) (1:1000; Cell Signaling).