Anti rhoa arh04

Antibodies were ordered from the following companies: anti-HIF1α (20960-1-AP, 1:1000), anti-HSF1 (51034-1-AP, 1:1000), anti-RhoC (10632-1-AP, 1:1000), and anti-Myc (60003-2-Ig, 1:2000) were from Proteintech (Chicago, IL,USA); anti-HSF1 phosphorylated at Ser326 antibody (ab76076, 1:2000) was from Abcam (Cambridge, MA, USA); anti-HA (#3724, 1:1000), and anti-HSP90α (#8165, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA); anti-RhoA (ARH04, 1:500) and anti-Rac1 (ARC03, 1:500) were from Cytoskeleton (Denver, CO, USA); anti-RhoB (sc-8048, 1:500) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Cdc42 (BA2442, 1:500) was from Wuhan Boster Biological Technology Co. Ltd. (Wuhan, China); and anti-Flag (F1804, 1:1000) was from Sigma-Aldrich. Cells were washed with PBS three times after treatment. Whole cell lysates were prepared using RIPA lysis buffer (Merck Millipore), with the addition of complete protease inhibitors (Roche). The protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific) and approximately 20 μg of cell lysates were used. Antibody binding was revealed using an HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Sigma-Aldrich). Antibody complexes were detected using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) and exposed in a Tanon-5200 machine.