Standardized Western Blotting Procedure

Western blotting was performed using standard gel electrophoresis procedure^{29 (link),43 (link),57 ,82 (link)}. 20 μg of lysed cell protein from each sample was run on 10% polyacrylamide gels, transferred onto polyvinylidene difluoride (PVDF) membranes, blocked with 5%(w/v) milk for 1 h. After washing, the membranes were incubated overnight at 4 °C in a solution of primary antibodies diluted in 5% w/v bovine serum albumin (BSA), 1X tris-buffered saline (TBS), 0.1% tween. Protein bands were visualized via horseradish peroxidase-conjugated secondary antibodies (1:5000, Cell Signaling) and ECL plus chemiluminescence kit (Amersham Biosciences, Piscataway, NJ) and scanned using C-DiGit blot scanner (Licor, Lincoln, NE). All blots derive from the same experiment and were processed in parallel.