Ab137875

Western blotting analysis was performed using standard techniques. The following antibodies were used: LSP1 (ab133506, Abcam), ERK1/2 (sc‐292838, Santa Cruz Biotechnology, Dallas, TX, USA), phosphorylated ERK1/2 (p‐ERK1/2; sc‐16982, Santa Cruz Biotechnology), cyclin D1 (ab137875, Abcam), B‐cell lymphoma 2 (Bcl2; ab32124, Abcam) and β‐actin (sc‐4778, Santa Cruz Biotechnology).

The BCPAP cells were lysed with RIPA lysis buffer (1% NP-40, 0.1% SDS, 50 mM DTT) containing protease inhibitor cocktail on ice. After centrifugation, the supernatant was collected in 1.5-ml centrifuge tubes. The cell lysate was loaded on 10% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels after being heated at 100 °C for 3 min for denaturation and then transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk at 37 °C for 2 h. The blocked membranes were washed with PBST buffer 2–3 times and incubated with the primary antibodies for 2 h at room temperature. After the membranes were washed four times with PBST buffer, they were incubated with a corresponding secondary antibody in PBST buffer at 4 °C overnight, followed by washing four times with PBST. The blots were detected using Enhanced Chemiluminescence Detection kit (KGP116, KeyGen BioTECH, Jiangsu, China). The primary antibodies used in the experiment were anti-p53 (ab31333, 1:1000), anti-CDK4 (ab137675, 1:2000), anti-cyclin D1 (ab137875, 1:5000) and p21 (ab109520, 1:1000), all purchased from Abcam; Anti-p-p53 (#9284, 1:1000) was purchased from CST; Anti- GAPDH (SC-365062, 1:800) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The secondary antibody used in the experiment were: goat anti-rabbit, goat anti-mouse IgG (1:4000 for both).