Figure 5
Sal B inhibits ET-1-induced ROCK II activation. (a) The expression of two isoforms of ROCK in rat HSCs. (b–c) ROCK I and ROCK II activation in response to ET-1 stimulation. Cells were treated with 10−8M ET-1 for various time intervals. ROCK I and ROCK II were precipitated using monospecific antibodies and were used for in vitro phosphorylation reactions with recombinant MYPT1(654-880) as a substrate. Phosphorylated MYPT1(654-880) was analyzed on 8% SDS-PAGE and detected by anti-Thr696-MYPT1 antibody. Immunoprecipitated ROCK I or ROCK II demonstrated the equal loading of proteins. (d) Cells were pretreated with 10−5M Y-27632, 10−5M Sal B, or with both for 30 min, then incubated with 10−8M ET-1 for 15 min. The mean density of Thr696-MYPT1(654-880) was normalized by that of ROCK II, and control samples were assigned a value of 1. Data are representative of three independent experiments. #P<0.01 versus control, *P<0.01 versus ET-1.
