Rabbit anti rhoa antibody

JIMT-1 cells were cultured on Matrigel in complete EBM-2 medium. After the medium was replaced by Hank’s balanced salt solution with DMSO or 0.5 μM salinomycin, the cells were cultured for another 2 h. Occasionally, 2 μg/mL Rho Activator II was added 30 min prior to the addition of 0.5 μM salinomycin. Cell lysates were prepared and subjected to GTP-bound Rho pulldown assays using an Active Rho Detection Kit (Cell Signaling Technology, Danvers, MA, USA) under the manufacturer’s instructions. RhoA was detected using rabbit anti-RhoA antibody (Cell Signaling Technology, #2117).

Purified mouse-anti human DLC1 [clone 3, cat. no. 612021, diluted 1:1000 for western blotting (WB)], was obtained from BD Biosciences. Rabbit anti-phospho-paxillin (Y118) antibody (cat. no. 69363S, diluted 1:400 for immunofluorescence, 1:1000 for WB), rabbit anti-phospho-MLC2 S19 antibody (cat. no. 3671S, 1:1000 for WB), rabbit anti-MLC2 antibody (cat. no. 8505S, 1:1000 for WB), rabbit anti-phospho-cofilin S3 antibody (cat. no. 3313S, 1:100 for WB), and rabbit anti-RhoA antibody (cat. no. 2117S, 1:1000 for WB) were purchased from Cell Signaling Technology. Mouse monoclonal anti-YAP1 antibody (63.7, cat. no. sc-101199, diluted 1:100 for immunofluorescence) was obtained from Santa Cruz Biotechnology. As a loading control, we used rabbit polyclonal anti-β-actin (Cell Signaling, Cat. no. 4967S, diluted 1:1000 for WB). To visualize F-actin we used PromoFluor 415-Phalloidin (cat. no. PK-PF415-7-01, diluted 1:200 for immunofluorescence) or PromoFluor 488-Phalloidin (cat. no. PK-PF488P-7-01, diluted 1:200 for immunofluorescence) from PromoKine. To visualize the nucleus, DAPI (Invitrogen, diluted 1:1000 for immunofluorescence) was used. Secondary antibodies coupled to Alexa Fluor 488 or 594 were from Invitrogen (diluted 1:250 for immunofluorescence). Secondary antibodies coupled to horseradish peroxidase (HRP) were obtained from Bio-Rad (diluted 1:1000 for WB).