Goat anti rabbit or goat anti mouse secondary antibody

Total protein was extracted from cells of each group using RIPA buffer. Denatured proteins were separated by SDS-PAGE through a 10% gel and then transferred to a PVDF membrane. The membrane was blocked with 5% milk or BSA for 2 hours and incubated with the appropriate primary antibody at 4°C overnight. The working concentrations of each antibody were as follows: anti-BMPER (1 : 500; Abcam, USA); anti-mechanistic target of rapamycin (mTOR), anti-phospho-mTOR, anti-LC3A/B, anti-Beclin-1, anti-PCNA, and anti-phospho-MEK 1/2 (1 : 1,000; CST, USA); anti-ERK1/2 (1 : 500; Bioss, Beijing); anti-phospho-ERK 1/2 (1 : 300; Bioss, Beijing); anti-MEK1/2 (1 : 300; Santa Cruz Biotechnology Inc.); anti-Bcl2, anti-Bax, anti-MMP2, anti-MMP9 (1 : 1,000; Proteintech, Rosemont, PA, USA); and anti-GAPDH (1 : 2,000; ZSGB- BIO, Beijing, China). Goat anti-rabbit or goat anti-mouse secondary antibody (1 : 3,000; ZSGB-BIO) was separately added and incubated for 2 hours after the membranes were rinsed with TBST. The protein bands were detected on a GDS8000 gel electrophoresis image analyzer (Thermo Fisher Scientific) using an enhanced electrochemiluminescence (ECL, Thermo Fisher Scientific) detection kit.

The procedure was performed as previously described [54 (link)]. Briefly, equal amounts of protein were subjected to 10% polyacrylamide gel electrophoresis and transferred to PVDF membrane. The membrane was incubated with rabbit anti-human PARP1 polyclonal antibody (Cell Signaling Technology, Danvers, MA, USA) or mouse anti-human β-actin monoclonal antibody overnight, followed by goat anti-rabbit or goat anti-mouse secondary antibody (Zsbio, Beijing, China). The bands were visualized using an ECL luminescent reagent (Millipore, Billerica, USA).