Figure 4

Inhibition of kinase activity by BDP5290 in cells. (A) MDA-MB-231 breast cancer cells expressing doxycycline inducible ROCK1, ROCK2 or MRCKβ kinase domains were established as indicated. Following treatment with doxycycline for 18 hours to induce expression, cell lysates were western blotted with antibodies as indicated. (B) Cells expressing doxycycline-induced MRCKβ, ROCK1 or ROCK2 kinases domains were treated with BDP5290 at indicated concentrations for 60 minutes prior to lysis and quantitative western blotting. (C) Inhibition of MLC phosphorylation by BDP5290 for each induced kinase domain. (D) Cells expressing doxycycline-induced MRCKβ, ROCK1 or ROCK2 kinases domains were treated with Y27632 at indicated concentrations for 60 minutes prior to lysis and quantitative western blotting. (E) Inhibition of MLC phosphorylation by Y27632 for each induced kinase domain. All results are shown as mean ± standard error of n = 4 independent replicates.