Fig. 1. ppMLC^T18/S19 is dependent on serum, Rho and Rho kinase. (A) Human fibroblasts were serum starved for 0 minutes, 2 minutes, 5 minutes and 15 minutes, and collected for analysis of ppMLC^T18/S19 by western blotting. (B) Cells were starved for 60 minutes, stimulated with 10% serum for 0 minutes, 1 minutes, 3 minutes and 10 minutes, and subjected to analysis of ppMLC^T18/S19. (C) Human fibroblasts, either serum starved or in 10% FBS, were treated with 10 µM Y-27632 or 25 µM ML-7 for 30 minutes, and collected in TCA for the analyses of MLC phosphorylation. DMSO (1:1000) was included as a vehicle control for ML-7. Similar results were obtained in three independent experiments. (D) Cells were treated with C3 exoenzyme/Lipofectamine mixture and incubated under serum-free condition for 8 hours. PBS/Lipofectamine was used for mock transfection. Cells were then either not stimulated or stimulated with 10% FBS for 2 minutes and collected in TCA for the analyses of ppMLC^T18/S19.