Rhoa antibody

RhoA activity was measured by Rhotekin assay in vSMC cells using Rhotekin RBD beads (Cytoskeleton Inc., Denver, CO) as described previously (Oinuma et al, 2012). Immunoblots were developed using RhoA antibody (Cell Signaling Technology, Beverly, MA).

The protein lysates (30–50 μg/sample assessed by BCA protein assay, Pierce Chemical Co., IL) were subjected to 10% SDS-Tris glycine gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were incubated with polyclonal GIT1 antibody (Cell Signaling, 1:1000), Rac1 antibody (Millipore 1:1000), Cdc42 antibody (Cell Signaling, 1:1000), RhoA antibody (Cell Signaling, 1:1000), MYO18A antibody (Santa Cruz. 1:1000), SNX27 antibody (Santa Cruz. 1:1000) and α-tubulin (Cell Signaling, 1:5000), at 4°c overnight. Peroxidase conjugated anti-rabbit antibody (1:5000) was used as a secondary antibody. The membranes were developed by Renaissance protein detection kit (DuPont NEN, MA, USA).

MDA-MB-231, MCF-10a, MDA-MB-157, MDA-MB-436, HMEC, and PC3 cells were purchased from Duke University Cell Repository, USA. LnCAP cells were a kind gift from Dr. Marie-Veronique Clement (National University of Singapore). HMEC cells were cultured in Clonetics™ MEGM™ Mammary Epithelial Cell Growth Medium (CC-3051). LnCAP and PC3 cells were maintained in RPMI complete media with 10% FBS and 1% Penicillin/Streptomycin (GIBCO, USA). MCF-10a cells were culture using DMEM-F12 (GIBCO, USA) supplemented with 10% FBS, 1% Penicillin/Streptomycin, 20 ng/ml EGF, 0.5 mg/ml Hydrocortisone, 10 μg/ml Insulin. The other cell lines were cultured in DMEM complete media with 10% FBS and 1% Penicillin/Streptomycin (GIBCO, USA). Matrigel inserts, plates, and growth factor-reduced Matrigel were purchased from BD Biosciences, USA. The monoclonal antibody against Gα13 (ST1629) was from Calbiochem, Germany, Gα12 antibody from Gentex (GTX114147) and antibody against α-tubulin (#010 M4813) was purchased from Sigma. Rho-A antibody was purchased from Cell Signaling (#2117). MicroRNA mimics (PremiRs) for miR-31, the PremiR-control, and antimiR-31 and the antimiR-control were purchased from Qiagen.