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. 2009 Oct 12;29(24):6488–6499. doi: 10.1128/MCB.01098-09

FIG. 6.

FIG. 6.

Cdk5 inhibition significantly decreases the Rho-GTP level and ROCK activity in spreading cells. (A) Cells were allowed to spread in the absence or presence of the Cdk5 inhibitor (olomoucine, 15 μM) or Rho inhibitor (C3 transferase, 2.0 μg/ml). Rho-GTP was isolated by affinity chromatography on Rhotekin beads and immunoblotted using a monoclonal anti-Rho antibody (upper panel). Total Rho level is shown in the lower panel. Lane 1, untreated; lane 2, treated with olomoucine; lane 3, treated with C3 transferase. (B) The results were quantified by densitometry, and the ratio of Rho-GTP to total Rho for each treatment was normalized to the untreated control. Statistical analysis demonstrated a significant decrease (P < 0.05) in Rho-GTP/total Rho in olomoucine-treated cells compared to that in untreated cells. C3 transferase was used as a positive control for inhibition of Rho. The values represent means ± SD (n = 3). (C) Cells were allowed to spread in the absence or presence of the Cdk5 inhibitor (olomoucine, 15 μM) or ROCK inhibitor (Y-27632, 10 μM) for 120 min. ROCK activity was assayed by in vitro kinase assay, using MBS of myosin phosphatase as a substrate. MBS(pT853) and total MBS were identified by immunoblotting using specific antibodies. Lane 1, untreated; lane 2, treated with olomoucine; lane 3, treated with Y-27632. (D) ROCK activity was expressed as the ratio of phospho-MBS [MBS(pT853)] to total MBS, normalized to untreated controls. The graph represents the average ratio of MBS(pT853)/MBS ± SD (n = 3). (E) Cells were transiently transfected with Cdk5 siRNA (siCdk5), replated after 48 h, and allowed to spread for 120 min. Rho-GTP was isolated by affinity chromatography on Rhotekin beads and detected by Western blotting using anti-Rho antibody (upper panel). Total Rho level is shown in lower panel. Lane 1, transfected with scrambled siRNA; lane 2, transfected with Cdk5 siRNA. (F) The results of immunoblotting experiments were quantified by densitometry. The values represent means ± SD (n = 3). Statistical analysis demonstrated a significant decrease (P < 0.01) in the Rho-GTP level in Cdk5 siRNA-transfected cells compared to that in scrambled siRNA-transfected cells.