P21waf1 cip1

Proteins were extracted by RIPA lysis buffer (Beyotime) with 1% phosphatase and protease inhibitors and separated by 10% SDS-PAGE (EpiZyme, Cambridge, MA, USA). Then, we transferred the proteins onto PVDF membranes (Pierce Biotechnology, Waltham, MA, USA) and blocked the membranes with 5% milk at 37 °C for 1 h. The membranes were incubated with primary antibody overnight at 4 °C. A subsequent 2 h incubation in secondary antibody was performed, and the membranes were then exposed to ECL reagents (Abcam, Cambridge, UK). Antibodies and dilutions were as follows: RAB20 antibody (Abcam, ab197209, 1:1000); α-tubulin (CST, #2144, 1:1000); β-actin (CST, #3700, 1:1000); CDK2 (CST, #2546, 1:1000); CyclinE1 (CST, #20808, 1:1000); CyclinD1 (CST, #55506, 1:1000); CyclinB1 (CST, #12231, 1:1000); cdc2 (CST, #9116, 1:1000); Chk1 (CST, #2360, 1:1000); cdc25C (CST, #4688, 1:1000); Phospho-cdc25C (CST, #4901, 1:1000); p53 (CST, #2527, 1:1000); Phospho-p53 (CST, #9286, 1:1000); and p21Waf1/Cip1 (CST, #2947, 1:1000).

By western blotting, the expression of various proteins in the miR-31 transfected and siRNA-treated NPC cells was detected. The antibodies against p21 Waf1/Cip1 (Abcam), Phospho-p53 (Ser15) (Abcam), HIF1α (Abcam), FIH1 (Santa Cruz), MCM2 (Santa Cruz) and ACTIN (Santa Cruz) were used.