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. 2010 Dec 8;286(8):6117–6127. doi: 10.1074/jbc.M110.167239

FIGURE 4.

FIGURE 4.

Inhibition of Rho kinase reduces LR11 phosphorylation in vivo. A, LR11 IPs from HEK293 cells transfected with V5-LR11 were incubated with kinase buffer-containing cold ATP, [32Pi]orthophosphate, and DMSO (Mock) or 10 μm Rho kinase inhibitor (RKI) for 30 min at 30 °C, as described under “Experimental Procedures.” Autoradiography indicated that RKI treatment inhibits LR11 phosphorylation in vitro. Immunoblotting identified V5-LR11 and ROCK2 in V5-LR11 IPs. B, HEK293 cells transfected with V5-LR11 were metabolically labeled with [32Pi]orthophosphate for 2 h in the presence of 50 μm RKI (RKI) or DMSO (Mock). LR11 was IPed with V5 antibody and subjected to SDS-PAGE. Autoradiography revealed that RKI reduced LR11 phosphorylation in vivo. Immunoblot data indicated that relatively equivalent amounts of LR11 were present in each IP. C, intensity of phosphoproteins at 250 kDa was quantified and normalized to the amount of LR11 in the IPs. Data shown in A and B are representative of three independent experiments.