Hypodermic needle pro

Whole-cell lysates were prepared in RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate) supplemented with 1mM PMSF, 1% SDS, and phosphatase inhibitors (1x PhosphoSTOP Phosphatase Inhibitor Cocktail, Roche Diagnostics, Cat#4906845001). Samples were lysed on ice for 20 min, sheared by passing cells through a G25 needle (B. Braun, Hypodermic Needle-Pro®, Cat#4658304), cleared by centrifugation (14000 x g, 15 min), and quanti ed using Pierce TM Bradford protein assay kit (Thermo Scienti c, Cat#23200). Lysates were boiled 5 min at 95° C with 6x reducing Laemmli buffer (0.12M Tris pH 6.8, 47% glycerol, 12% SDS, 0.6M DTT, 0.06% bromophenol blue). Samples were separated on a polyacrylamide gel and transferred to PVDF membranes using the mini wet/tank blotting system (Bio-Rad). After blocking with 5% BSA in Tween-20/PBS, membranes were probed with primary antibodies prepared in blocking solution overnight at 4° C on a roller, followed by incubation with horseradish peroxidase-conjugated secondary antibody in blocking solution for 1 h at room temperature and ECL detection (Thermo Fisher, Cat#34096) by the ChemiDoc XRS+ system (Bio-Rad). Primary and secondary antibodies used for Western blotting are listed in Supplementary Table S1. Quantitative analysis of protein expression relative to GAPDH was done using Image Lab software (Bio-Rad).