Blots were incubated overnight in 1 X PBS containing 0.05% Tween 20 and 5% milk with the primary antibody at 4°C. The following primary antibodies (1/50 - 1/500 dilution) were utilised: anti-human ROCK-1 mouse polyclonal primary antibody (G-6 sc17794), ROCK-2 rabbit polyclonal G6 (sc5561), RhoA mouse monoclonal (26C4 sc418), caveolin-1 rabbit polyclonal (N-20 sc894), Rho8 (Rnd3) mouse monoclonal (clone 4 sc53874), moesin goat polyclonal (C-15 sc6410), pmoesin Thr558 goat polyclonal (sc12895), OTR rabbit polyclonal (H-60 sc33209), RhoGDI rabbit polyclonal (A20 sc360), MYPT 1 rabbit polyclonal (H-130 sc25718), MYL-9 (MLC) (D-15 sc-34487), ppMLC 18/19 goat polyclonal (sc12896 Santa Cruz Biotechnology Inc., Rockford, IL, USA), pMYPT 853 rabbit (Upstate, Millipore, Billerica, MA, USA), pMYPT Thr696 rabbit polyclonal (Upstate Millipore, USA ABS454), MLC mouse monoclonal (ab97981 Abcam plc, Milton Road, Cambridge, UK), RhoE (Rnd3) mouse monoclonal 3664 (Cell Signaling Technology, Danvers, MA, USA), caspase 3 monoclonal rabbit (Cell Signaling, USA 8G10 9665), cleaved caspase 3 rabbit monoclonal (Cell Signaling, USA Asp175 5A1E), calponin mouse monoclonal (DakoCytomation Ltd, UK M3556), pMAPK mouse monoclonal antibody to ERK1 and 2 pT185 and Y187 (Abcam, UK 50011) or 1/10,000 dilution of β-actin (ACTB) mouse monoclonal AC-15 (Sigma-Aldrich, Ireland) antibodies. Blots were washed and incubated in either 1/1,000 dilution of donkey anti-goat (Pierce Technology, Hallandale, FL., USA), 1/1000 goat anti-rabbit (Pierce Technology, USA), or 1/5000 dilution anti-mouse (Millipore, USA) horseradish peroxidase-conjugated secondary antibodies, the bound secondary antibody was detected as previously described. The membranes were then scanned with the fluorescence imager (FluorchemTM 8900, Alpha Innotech Corporation, San Leandro, California, USA) and AlphaEaseFC software was used to detect the signal, the image was processed and protein expression levels were determined by densitometric analysis compared to corresponding levels of the housekeeping protein, β-Actin [
8].