Ab227443

The western blot assay was carried out as previously described (Zhao et al., 2021 (link)). The primary antibodies against caspase-3 (Abcam, ab65080), cyclin D1 (Abcam, ab226977), CDK4 (Abcam, ab137675), p21 (Abcam, ab227443), FPN (Abcam, ab235166), TF (Abcam, ab84036), FTH1 (Abcam, ab65080), GPX4 (Abcam, ab125066), Nrf2 (Abcam, ab137550), HO-1 (Abcam, ab13243) and β-actin (Abcam, ab8227) were used in this investigation.

The total protein from the tumor samples or cells was extracted using a cell protein extraction kit (Thermo, USA). The bicinchoninic acid (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) method was used to determine the total protein concentration and to perform a quantitative detection of the samples. A sodium lauryl sulfate-polyacrylamide gel was prepared, and 50 µg of protein sample was added to each well. After subjecting the samples to 75 V constant voltage electrophoresis for 30 min, 100 V constant voltage electrophoresis was performed for 2 h. The wet transfer method was applied to electrically transfer the protein to a polyvinylidene fluoride membrane. After blocking the membrane with 5% bovine serum albumin for 60 min, the corresponding primary antibody was added and incubated overnight at 4 °C. Next, a secondary antibody (goat anti-rabbit IgG H&L (HRP) preabsorbed, 1/1000; ab7090; Abcam, Cambridge, MA, USA) was incubated with the membrane for 1 h at room temperature. Using an Odyssey two-color infrared laser imaging system, the membrane was scanned. β-actin was used as an internal control to analyze the level of target protein expression. The primary antibodies included CARM1 (1/5,000; ab245466; Abcam), CCND2 (1/500; ab230883; Abcam), P21 (1/500; ab227443; Abcam) and β-actin (1/5,000; ab179467; Abcam).

Cells were lysed in lysis buffer (30 mM Tris‐HCl, 150 mM NaCl, 1% NP‐40, and 0.1% SDS; pH 7.4) supplemented with a protease inhibitor cocktail (Roche). The BCA Protein Quantitation Kit (Genescript) was used to determine protein concentration. Proteins were separated using 10% SDS‐PAGE and blotted electrophoretically onto polyvinylidene difluoride (PVDF; Immobilon) membranes of 0.45 µm pore size. Membranes were blocked with 5% bovine serum albumin (BSA) in phosphate‐buffered saline containing 0.1% tween‐20 (PBST) for 1 hour at room temperature, followed by incubation with primary antibody overnight at 4°C: anti‐p21 antibody (ab227443, Abcam), anti‐p16 antibody (ab151303, Abcam), anti‐bcl‐2 antibody (ab59348, Abcam), anti‐bax antibody (ab53154, Abcam), anti‐PTEN antibody (ab31392, Abcam), anti‐Akt antibody (ab8805, Abcam), anti‐pAkt antibody (ab18206, Abcam), and anti‐GAPDH antibody (ab9485, Abcam). HRP‐linked goat anti‐rabbit IgG secondary antibodies (Amersham) were incubated with the membranes for 1 hour at room temperature in PBST containing 5% BSA, followed by chemiluminescent detection. A C‐DiGit Blot Scanner and Super Signal West Femto Maximum Sensitivity Substrate Kit (provided by Thermo) were used to detect bound antibodies.