Anti gef h1

A monoclonal mouse anti-ASAP1 antibody was purchased from Abnova. A polyclonal rabbit anti-ASAP1 serum was raised in our lab as previously described (31 (link)). Anti-paxillin and anti-GM130 antibodies were purchased from BD Biosciences, anti-ROCK2 antibody from BD Transduction Laboratories, anti-HABP4 antibody from Bioss USA, anti-Akt, anti-Arf6, anti-GEF-H1, anti-JNK2, anti-PRKAR1A (PKA RI-α/β), anti-ROCK1, anti-SMARCA5 (SNF2H), anti-TIAM1, and anti-VAV2 from Cell Signaling Technology, anti-β-COP antibody from Invitrogen, IRDye680 anti-mouse IgG and IRDye800 anti-rabbit IgG were from LI-COR, and anti-Brag2 (IqSec1) antibody was from Sigma Precision Antibodies.

Cells were washed once with ice-cold PBS and lysed in ice-cold RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1× protease inhibitor cocktail, and 1× phosphatase inhibitor cocktail). Cell debris was removed by centrifugation at 14,000 g for 15 min at 4°C. Protein concentration in the supernatants was determined via Bradford assay. Equal amounts of total protein were mixed with 5× Laemmli sample buffer, boiled at 95°C for 10 min and separated by SDS-PAGE. After electrophoresis, proteins were transferred on a PVDF membrane using a semidry blotter. Blots were blocked for 30–60 min at RT with 5% BSA in TBS-T (20 mM Tris, pH 7.6, 137 mM NaCl, and 0.1% Tween-20) and incubated overnight at 4°C with primary antibodies anti–GEF-H1 (1:200; 55B6; Cell Signaling), anti-Ect2 (1:500; C-20; Santa Cruz Biotechnology), and anti–α-Tubulin (1:20,000; clone B-5-1-2; Sigma-Aldrich) in blocking solution. Membranes were washed three times with TBS-T and incubated with HRP-conjugated anti–mouse (for anti–α-Tubulin) or anti–rabbit (for anti–GEF-H1) secondary antibodies (1:20,000) for 1 h at RT. After additional washing steps with TBS-T and TBS (20 mM Tris, pH 7.6, and 137 mM NaCl), proteins were visualized with ECL Western blotting substrate.