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

FIG. 3.

FIG. 3.

Dominant negative Cdk5 and Cdk5 siRNA significantly decrease MRLC phosphorylation. (A) Cells were transfected with GFP-tagged constructs of wild-type Cdk5 (GFP-Cdk5) or dominant negative Cdk5(D144N) (GFP-dn-Cdk5). After 48 h incubation for expression of the transfected construct, the cells were replated and allowed to spread for 2 h, and proteins were extracted and immunoblotted with specific antibodies to detect pMRLC (upper panel), total MRLC (middle panel), and tubulin (lower panel). Lane 1, untransfected; lane 2, transfected with GFP-Cdk5; lane 3, transfected with GFP-dnCdk5(D144N). (B) Results of three independent experiments of the type shown in panel A were quantified by densitometry, and the ratio of pMRLC to MRLC was plotted to determine the relative levels of pMRLC. The values represent means ± SD. (C) Cells were transiently transfected with Cdk5 siRNA (siCdk5) or scrambled siRNA, replated after 48 h, and allowed to spread for 120 min. Cdk5 was detected by immunoblotting with anti-Cdk5 monoclonal (J-3) antibody (upper panel). Tubulin was used as a loading control (lower panel). Lane 1, untransfected; lane 2, transfected with scrambled siRNA; lane 3, transfected with Cdk5 siRNA. (D) The results of immunoblotting experiments (n = 3) were quantified by densitometry and normalized to tubulin. The values represent means ± SD. Endogenous Cdk5 was significantly lower (P < 0.01) in Cdk5 siRNA-transfected cells than in untransfected cells or cells transfected with scrambled siRNA. (E) Experimental conditions were the same as in panel C. Levels of pMRLC, total MRLC, and tubulin were determined by immunoblotting. Lane 1, untransfected; lane 2, transfected with scrambled siRNA; lane 3, transfected with Cdk5 siRNA. (F) Results of three independent experiments of the type shown in panel E were quantified by densitometry, and the ratio of pMRLC to MRLC was plotted to determine relative levels of pMRLC. The values represent means ± SD. Statistical analysis demonstrated a significant (P < 0.01) decrease in pMRLC level in Cdk5 siRNA-transfected cells compared to that in untransfected or scrambled siRNA-transfected cells.