Western Blot Analysis of Protein Lysates

The detailed procedure was carried out as previously described^{47 (link)}. The harvested cells were lysed in ice-cold RIPA buffer with protease inhibitors. The protein lysates were separated on SDS–PAGE and then transferred to PVDF membranes (Roche Diagnostics). After blocked for 2 h in 5% bovine serum albumin (BSA) in 1 × TBS-T (0.5% Tween-20), the membranes were then incubated at 4 °C overnight with the indicated primary antibodies. Horseradish peroxidase (HRP)-conjugated secondary antibodies along with an ECL kit (GE Healthcare/Amersham Pharmacia Biotech, #32106) were used to detect protein signals. Multiple exposures were taken using the Western Bright ECL detection system (Advansta, CA) to select images within the dynamic range of the film (GE Healthcare Amersham Hyperfilm ECL, #28906838). Signals were normalized to GAPDH bands. The dilution ratio of antibodies for western blot analysis were shown in Supplementary Table 3 (the fourth column). All blots derive from the same experiment and were processed in parallel. Antibody information was listed in Supplementary Table 3.