P115 rhogef

Cell lysates were harvested in RIPA buffer supplemented with HALT protease inhibitor and stored at -20°C. Proteins were separated on a 4–12% SDS-PAGE gel and blotted on PVDF membranes. Immunoblots were performed using antibodies directed against HA (sc-7392, Santa Cruz Biotechnology), p115 RhoGEF (sc-74565, Santa Cruz Biotechnology), ROCK1 (sc-5562, Santa Cruz Biotechnology), c-Src (sc18, Santa Cruz Biotechnology), β-actin-HRP (sc-47778, Santa Cruz Biotechnology), endothelial cell growth factor receptor (EGFR; Cell Signaling; D3881), Streptavidin-HRP (Thermo Scientific; 21130), UL44 (CA006-100, Virusys), and TurboID (AS204440, Agrisera) and if required, with the appropriate HRP conjugated secondary antibodies (anti-mouse sc-25409 and anti-rabbit sc-2357).

Collagen-1 was purchased from either BD Biosciences (Bedford, MA) or Invitrogen (Carlsbad,CA). E-cadherin antibody was obtained from BD Biosciences. p115RhoGEF, LARG and PRG antibodies were purchased from Santa Cruz (Santa Cruz, CA). Antibody for β-actin was purchased from Cell Signaling (Danvers, MA). Monoclonal anti-vimentin antibody (clone V9) was purchased from Sigma-Aldrich, ZO-1 antibody was purchased from Invitrogen (Carlsbad,CA), hDia1(human) antibody was purchased from Proteintech (Chicago, IL). Lipofectamine 2000 and RNAiMAX was purchased from Invitrogen. siRNA oligonucleotides (p115RhoGEF: CATACCATCTCTACCGACG, LARG: GAAACTCGTCGCATCTTCC and PRG: ACTGAAGTCTCGGCCAGCT) [41] (link) were synthesized by Dharmacon. ON-TARGETplus Non-targeting siRNA was purchased from Dharmacon. siGENOME SMARTpool siRNAs library to target RhoA effector genes [27] (link) and individual siRNAs to target DIAPH1: DIAPH1 oligo#1 GAAGUGAACUGAUGCGUUU and oligo #2 GAUAUGAGAGUGCAACUAA were purchased from Dharmacon. pQCXIB-CMV/TO-DEST (w320) created by Dr Eric Campeau and pBabe-puro-Rho biosensor (created by Klaus Hahn) were obtained from Addgene. myc-DEST was a gift from Dr Adi Dubash.