Anti cdkn2c

Protein lysates of ESCC cells were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22-mm NC membranes (Sigma), and incubated with specific antibodies: anti-CDK6, anti-CDK4, anti-CDK2, anti-cyclinD1, anti-cyclinD3, anti-p27, anti-p21, anti-CDKN2C (Abcam, Shanghai, China), anti-EZH2, anti-EED, anti-SUZ12, anti-EZH1, and anti-β-actin (Cell Signaling Technology). The dilution ratio of the primary antibodies was 1:1,000, although anti-β-actin was diluted to 1:8,000 for Western blotting. Protein bands were visualized with Super Signal Chemiluminescence Substrate (Thermo Scientific) and β-actin was used as a control.

Human PCa cell lines (PC3 and C4-2) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) with 10% fetal bovine serum (FBS, Cat# 10270-106, Gibco, UK) and penicillin–streptomycin combination solution (10 kU/mL penicillin and 10 mg/mL streptomycin, Cat# PB180120, Procell, Wuhan, China) at 37 °C and 5% CO₂. PCa cells were transfected with small interfering RNAs (siRNAs) (General biological system, Anhui, China) using Lipofectamine 3000 reagent (Lot# 2307436, Invitrogen, USA). The sequences of si-cyclin-dependent kinase inhibitor 2C (si-CDKN2C), si-Rac GTPase-activating protein 1 (si-RACGAP1), and the nontargeting control (NC) are shown in Additional file 1: Table S1. The antibodies used were as follows: anti-CDKN2C (Cat# ab192239, Abcam, Cambridge, UK), anti-RACGAP1 (Cat# ab134972, Abcam, Cambridge, UK), anti-β‐actin (Cat# AF7018; Affinity, OH, USA), and anti-β‐tubulin (Cat# AF7011, Affinity, OH, USA). The secondary antibodies included goat anti‐rabbit for CDKN2C, RACGAP1, and β-actin (Cat# AS014, ABclonal, Wuhan, China) and goat anti-mouse for β-tubulin (Cat# S0002, Affinity, OH, USA).