Anti cdc42

The whole-cell extract was obtained in RIPA buffer in the presence of standard protease and phosphatase inhibitors. The protein content was determined with a protein assay reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA), using bovine serum albumin as a standard. Equal protein content of total cell lysates was resolved on polyacrylamide gel (Bolt 4–12% Bis-Tris Plus Invitrogen, Carlsbad, CA, USA), electro-transferred to PVDF membranes (iBlot Invitrogen, Carlsbad, CA, USA) and incubated with specific primary antibodies: anti-YAP (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; sc #101199; dil.1:1000); anti-p-YAP (Cell Signaling Technology, Inc., Beverly, MA, USA; #13008; dil.1:1000); anti-β Actin (MP Biomedicals, Santa Ana, CA, USA; # 69100; dil.1:10000); anti-Rac1, anti-RhoA and anti-Cdc42 (all Cell Biolabs, San Diego, CA, USA; STA-405; dil.1:500); anti-Zeb1 (Abcam, Cambridge, MA, USA; #180905; dil.1:2000); anti-Snail1 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; sc-271977; dil.1:1000); anti-GSN (Sigma-Aldrich, St Louis, MO, USA; G-4896, dil.1:1000). Membranes were developed using ECL detection reagents (GE Healthcare Life Sciences, Uppsala, Sweden) on a UVITEC imaging system (UVITEC, Cambridge, UK) or ChemiDocMP system (Bio-Rad Laboratories Inc.). Quantitative analysis of western blots was performed using ImageJ (NIH) software [23 (link)].

RhoA, Rac1, and Cdc42 activity assays were performed using the appropriate assay kits according to the manufacturer’s instructions (Cell Biolabs, San Diego, CA, USA). The relative levels of active GTPases were determined by measuring the amount of GTPases sedimented by the GST-Rac1/Cdc42-binding domain of p21-activated kinase 1 or the GST-RhoA-binding domain of rhotekin relative to the amount in whole-cell lysates. Bound proteins were resolved on 15% SDS-PAGE gels and immunoblotted with anti-RhoA (1:1000), anti-Rac1 (1:500), or anti-Cdc42 (1:1000) antibodies (Cell Biolabs).