Membranes were blocked with 5% skimmed milk in Tris Buffered Saline (TBS), followed by incubation with specific anti-phospho-protein primary antibody (typically 1:1000 dilution) in TBS with 1% skimmed milk and 0.1% Tween-20 (TBS-T), overnight at 4°C. This was then followed by washes in TBS-T and the application of appropriate HRP-conjugated secondary antibody (typically 1:5000 dilution) for 1hr at room temperature, followed by further washes in TBS-T. proteins were visualised with Super-Signal West Femto ‘Phospho’ Substrate (Thermo scientific). Membranes were then stripped in Restore western blot stripping buffer (Thermo Scientific), re-blocked and re-incubated with corresponding antibody and appropriate secondary antibodies, as above. proteins were ‘total’ ‘Total’ visualised with either ECL plus or ECL prime (Amersham, GE healthcare). All images were captured and quantified using the ChemiDoc XRS+ gel imaging system (Biorad). Changes in protein expression were determined as a ratio of target protein signal, over signal for the protein GAPDH, acting as a loading control for each sample. An ‘housekeeping’ estimate of the amount of target protein that was phosphorylated in response to acute treatments was calculated both as a ratio of over signal for each protein ‘phospho’ ‘total’ sample, to give an indication of the of each protein that is phosphorylated in response fraction to BK, and as a ratio of over GAPDH, to reveal the influence of TGF- and ‘phospho’ ARHGEF1 siRNA on phosphorylation of each protein, in addition to the acute effect of total BK. The effects of treatments on these ratios were then expressed as a percentage of control (no BK, no TGF-, run on the same gel). Antibodies were obtained from: Cell Signalling (anti-phospho-Src Materials and Reagents. (tyr416), anti-Src, anti-phospho-MLC (ser19), anti-MLC, anti-MYPT1, anti-p115RhoGEF,
Membranes were blocked with 5% skimmed milk in Tris Buffered Saline (TBS), followed by incubation with specific anti-phospho-protein primary antibody (typically 1:1000 dilution) in TBS with 1% skimmed milk and 0.1% Tween-20 (TBS-T), overnight at 4°C. This was then followed by washes in TBS-T and the application of appropriate HRP-conjugated secondary antibody (typically 1:5000 dilution) for 1hr at room temperature, followed by further washes in TBS-T. ‘Phospho’ proteins were visualised with Super-Signal West Femto Substrate (Thermo scientific). Membranes were then stripped in Restore western blot stripping buffer (Thermo Scientific), re-blocked and re-incubated with corresponding ‘total’ antibody and appropriate secondary antibodies, as above. ‘Total’ proteins were visualised with either ECL plus or ECL prime (Amersham, GE healthcare). All images were captured and quantified using the ChemiDoc XRS+ gel imaging system (Biorad). Changes in protein expression were determined as a ratio of target protein signal, over signal for the ‘housekeeping’ protein GAPDH, acting as a loading control for each sample. An estimate of the amount of target protein that was phosphorylated in response to acute treatments was calculated both as a ratio of ‘phospho’ over ‘total’ signal for each protein sample, to give an indication of the fraction of each protein that is phosphorylated in response to BK, and as a ratio of ‘phospho’ over GAPDH, to reveal the influence of TGF- and ARHGEF1 siRNA on total phosphorylation of each protein, in addition to the acute effect of BK. The effects of treatments on these ratios were then expressed as a percentage of control (no BK, no TGF-, run on the same gel). Materials and Reagents. Antibodies were obtained from: Cell Signalling (anti-phospho-Src (tyr416), anti-Src, anti-phospho-MLC (ser19), anti-MLC, anti-MYPT1, anti-p115RhoGEF,