Odyssey clx analyzer

Cells were harvested, washed in PBS and lysed for 5min on ice in RIPA buffer (25mM Tris pH 7.5, 150mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% NP40 analog, 1x protease (Cell Signaling) and 1:500 Universal Nuclease (ThermoFisher Scientific). Protein concentration was determined from total cell lysates using DC protein assay (Biorad). Gel electrophoresis was done on Novex Tris-Glycine gels (ThermoFisher Scientific) before transfer using the Trans-Blot Turbo blotting system and nitrocellulose membranes (Biorad). All immunoblotting was performed in Intercept Protein blocking buffer (Licor). Washes were done in TBS + 0.1% Tween-20 (Sigma). Specific primary antibodies were diluted 1:100–1:5000 in blocking buffer. Fluorescent-coupled secondary antibodies were diluted 1:10,000 in blocking buffer. Membranes were imagined with an Odyssey CLx analyzer (Licor) or by chemiluminescence. In a few instances, the same lysates were loaded on parallel gels to avoid antibody cross-reactivity, or because the proteins of interest could not be resolved on the same percentage gels. In these cases, a loading control is provided for all gels, and panels from the same immunoblots are connected with a dotted line on the figure. All raw immunoblots pictures are provided in the supplemental information document.