Figure 2

Dose response of phospho-MLC to Y27632 concentration. A) MLC diphosphorylation decreased with increasing concentration of Y27632 as shown by representative histograms of the range of phospho-MLC fluorescence in 150 cells measured. Histograms labels refer to concentration (μM) of Y27632 added. B) Percentage inhibition of MLC-phosphorylation obtained by quantitative light microscopy and by Western blotting, plotted as a function of Y27632 concentration. The curves were fit to an exponential function as described in Materials and Methods to obtain an IC₅₀ of 2.1 ± 0.6 μM by quantitative imaging and 1.3 ± 1.2 μM by Western blotting. Error bars indicate standard deviations of 3 replicate experiments. Representative images of the western blots used to quantify phospho-MLC, total MLC, and the concentrations of Y27632 used are shown below the graph.