Transblot turbo lf pvdf

Cells were washed with ice-cold PBS and lysed with 25 mM Hepes, pH 7.2 (H4034; Sigma-Aldrich), 125 mM potassium acetate (104820; Merck Millipore), 2.5 mM magnesium acetate (105819; Merck Millipore), 5 mM EGTA (E3889; Sigma-Aldrich), 0.5% Triton-X-100 (Sigma-Aldrich) and 1 mM dithiothreitol (DTT; D0632; Sigma-Aldrich) supplemented with protease inhibitor cocktail (P9340; Sigma-Aldrich) or lysed in 2× sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 200 mM DTT and 0.004% bromophenol blue). Lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% or 4–20% gradient gels (mini-PROTEAN TGX; Bio-Rad). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (TransBlot® Turbo^TM LF PVDF, Bio-Rad) followed by antibody incubation in 2% bovine serum albumin in Tris-buffered saline with 0.1% Tween-20. Membranes incubated with fluorescent secondary antibodies (IRDye680 or IRDye800; LI-COR) were developed with an Odyssey infrared scanner (LI-COR), whereas those incubated with horseradish peroxidase-conjugated antibodies were developed using Clarity Western ECL substrate solutions (Bio-Rad) with a ChemiDoc XRS+ imaging system (Bio-Rad). Quantification of immunoblots was done using the Odyssey Software. Please see Supplementary Figs. 9–11 for uncropped membranes.

Cells were washed twice with ice cold PBS and lysed with 2 x sample buffer (125 mM Tris-Cl, pH 6.8 [Sigma, T1378], 4% SDS [Sigma, L-5750], 20% glycerol [Sigma, G5516], 200 mM DTT [Merck, D0632], 0.004% bromophenol blue) supplemented with protease inhibitor cocktail (cOmplete, EDTA free; Roche, 05056489001) and for signaling experiments supplemented with phosphatase inhibitors (phosSTOP; Roche, 04906837001). Lysates were subjected to SDS-gel electrophoresis on 10% or 4-20% gradient gels (mini- or midi-PROTEAN TGX; Bio-Rad, 5671095, 5671035, 5671034, 5671094, 4561036, 4561095). Proteins were transferred to PVDF membranes (TransBlot® Turbo^TM LF PVDF; Bio-Rad, 170-4275, 170-4274) followed by blocking and antibody incubation in 2% BSA (Merck, BSAV-RO 10735094001) in Tris-buffered saline (pH 7.5), with 0.05% Tween-20 (Sigma, P1379). Membranes incubated with fluorescent secondary antibodies (LI-COR) were developed using an Odyssey infrared scanner (LI-COR), or incubated with horseradish peroxidase-conjugated antibodies and developed using a Clarity Western ECL substrate solution (Bio-Rad, 1705060) and ChemiDoc XRS+ imaging system (Bio-Rad). Quantification and analysis of immunoblots was performed with Odyssey Software (version 3.0.30) or ImageJ (version 1.52p).