Figure 5
Specificity of phospholipase C-β phosphorylation.32P-Labeled NG108–15 cells were incubated with drugs. A:Lane 1, no addition. Lane 2, 100 nm DPDPE for 1 min. Lane 3, as forlane 2 but incubated for 10 min with 10 μmnaloxone. Lane 4, 1 μm LPA acid for 1 min.Lane 5, 100 nm DPDPE and 1 μmLPA together for 1 min. Lane 6, 1 μmbradykinin for 1 min. Lane 7, as for lane 2 but preincubated with 10 μm U73122. Lane 8, 10 μm U73122 for 10 min. C: Lane 1, no addition. Lanes 2and3, 100 nm DPDPE for 1 and 2 min, respectively. Lanes 4and5, 1 μm LPA for 1 and 2 min, respectively. Cells subsequently were lysed and immunoprecipitated with anti-PLC-β3 antibody (A) or anti-PLC-β1 (C), and samples were subjected to SDS-PAGE and autoradiography and visualized with a Molecular Dynamics PhosphoImager. Result shows a representative experiment repeated four times. B, Quantification of PLC-β3 phosphorylation of experiment shown in A; results are mean ± standard deviation of four independent experiments.