Anti rabbit and anti mouse antibodies

For Western blotting, protein lysates were prepared in equivalent volumes to contain 20 μg of total protein and mixed with Laemmli Buffer (Bio-Rad) containing either TCEP or β-mercaptoethanol and boiled for 5 minutes at 95°C to reduce and linearize protein. After cooling, samples were loaded onto 4–20% gradient gels (Bio-Rad) and separated by SDS-PAGE. Proteins were transferred to 0.2 μm PVDF membranes using the Trans-blot Turbo transfer apparatus (Bio-Rad). Following the transfer, the membranes were blocked with 5% non-fat milk or bovine serum albumin (BSA) dissolved in 10 mM Tris-buffered saline, 0.1% Tween-20 (TBS-T) for 90 minutes at room temperature. The membranes were incubated with primary antibodies against (human) proteins of interest at dilutions indicated in S1 Table overnight (16 h) at 4°C with orbital shaking. Membranes were washed with TBS-T buffer and incubated with horseradish peroxidase-conjugated secondary antibodies against the species; the primary antibodies were raised for 1 h at room temperature with orbital shaking. Anti-rabbit and anti-mouse antibodies were diluted at 1:3000 (Cell Signaling), and the anti-goat antibody was diluted 1:1000. Proteins of interest were visualized by chemiluminescent HRP detection (Bio-Rad). Primary and secondary antibodies were diluted in the same buffer used for blocking.