329063.fig.001a
(a)
329063.fig.001b
(b)
329063.fig.001c
(c)
329063.fig.001d
(d)
Figure 1: PKCε phosphorylates and binds to RhoA. (a) PKCε phosphorylates RhoA. Recombinant RhoA was incubated with or without recombinant PKCε in kinase buffer containing PKC activators, phosphatidylserine and diacylglycerol, and [32P]ATP for 30 minutes at 25°C. Subsequently, the incubation reaction was terminated, separated by SDS-PAGE and visualized using autoradiography. (b) Pro-Q Diamond staining of phosphorylated RhoA. Recombinant RhoA was incubated with or without recombinant PKCε in kinase buffer containing PKC activators, phosphatidylserine and diacylglycerol, and ATP for 30 minutes at 25°C. Subsequently, the incubation reaction was terminated, separated by SDS-PAGE and visualized using a stain specific to phosphoproteins. (c) PKCε binds to RhoA. Recombinant PKCε was incubated with recombinant RhoA in kinase buffer containing PKC activators, phosphatidylserine and diacylglycerol, for 30 minutes at 25°C. The binding reaction was immunoprecipitated using agarose-conjugated anti-PKCε or nonspecific IgG antibody. The immunoprecipitated proteins were visualized by western blot analysis using an anti-RhoA antibody. Two independent immunoprecipitation experiments are presented. (d) The kinase domain of PKCε binds to RhoA. Recombinant His-tagged PKCε-kinase domain was incubated with recombinant GST tagged-RhoA in kinase buffer containing PKC activators, phosphatidylserine and diacylglycerol, for 30 minutes at 25°C. The binding reaction was immunoprecipitated using agarose-conjugated anti-His, anti-RhoA, or nonspecific IgG antibody. The immunoprecipitated proteins were visualized by western blot analysis using an anti-His and anti-RhoA antibody.