Western Blot Protein Detection Protocol

Protein lysates (40 μg per lane) were loaded on a 4–15% SDS–PAGE gel (Bio-Rad), separated in 1X Tris-Glycine Buffer (Bio-Rad), and transferred to Polyvinylidene fluoride (PVDF) membranes (Millipore) via a wet electro-blotting system (Bio-Rad), all according to the manufacturer’s directions and as described previously (109 (link)). PVDF membranes were blocked for 1 h in 5% non-fat milk (Bio-Rad) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T; Bio-Rad), then incubated overnight at 4°C with antibodies listed in Table S2 in 5% BSA in TBS-T. Blots were then probed with anti-Rabbit IgG conjugated with horseradish peroxidase (1:5,000; Cell Signaling Technology) in 5% BSA in TBS-T for 1 h at room temperature. Signal was detected with the Immobilon Western Chemiluminescent HRP Substrate (Millipore), and blot images were collected with a Bio-Rad ChemiDoc imaging system.