Western Blotting Analysis

Total proteins were isolated from CC cells, which were lysed on ice with a RIPA lysis buffer (Cell Signaling Technology, MA). The protein concentration was determined using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Extracted protein (30 µg) was separated by 12% SDS-PAGE and transferred onto a PVDF membrane (GE Healthcare Life Sciences, Piscataway, NJ). The membranes were blocked with 5% non-fat milk for 1 h and then incubated with anti-HK2 antibody (1:1000, #2106, Cell Signaling) and anti-GAPDH antibody (1:5000, #2118, Cell Signaling) at 4°C overnight. Then, the membranes were subsequently incubated with secondary antibodies for 2 h at room temperature. The protein bands were detected using an ECL detection kit (Amersham Pharmacia Biotech, UK).