Immunoblotting Protocol for Protein Analysis

Immunoblotting was performed as described previously [23 (link), 24 (link)]. In brief, cells were treated with the indicated drug and lysed. Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes that were then blocked with 5% skimmed milk in TBS-Tween buffer (50 mM Tris-HCL [pH 7.4], 150 mM NaCl, and 0.05% Tween 20). Immunoblotting was carried out using primary antibodies (Table S2) appropriately diluted in TBS-Tween buffer containing 5% BSA and 0.05% sodium azide. Signals were detected with the appropriate horseradish peroxidase-conjugated secondary antibodies appropriately diluted in TBS-Tween buffer. Images were visualized with a LAS-4000 mini-image analyzer (FUJIFILM, Tokyo, Japan) and analyzed with MultiGauge software (FUJIFILM).