Figure 10.

Expression of wild-type Rac1 in OCRL-depleted cells rescues enhanced phosphorylation levels of cofilin. (A) HeLa cells were treated with siRNA against OCRL, followed by transfection with constructs expressing myc-tagged Rac-wt or RacN17, prior to stimulation with 10 ng/ml EGF for 20 min, fixation and immunofluorescence analysis. Rac1 variants were detected by staining cells with an anti-myc antibody, followed by an AlexaFluor546-conjugated secondary antibody (red). Phospho-cofilin was visualized using an anti-phospho-cofilin antibody, followed by an AlexaFluor488-conjugated secondary antibody (green). * indicates cells transfected with a Rac1 expression vector. Scale bar = 10 µm. To exclude off-target effects, three different siRNAs against OCRL were independently applied. (B) Quantification of the mean fluorescence of phospho-cofilin-stained cells was performed by the Metamorph software. To enable direct comparison, all images were taken under the same magnification and laser intensity settings. The fluorescence intensity of OCRL-depleted cells expressing either Rac-wt or RacN17 is expressed as percentage of cells depleted of OCRL without Rac1 re-expression. Results are from three independent experiments with 10 sections per condition of each experiment evaluated and are shown as the mean ± SD. **P = 0.003 (two-tailed Student's t-test).