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. 2012 Aug 19;21(23):5019–5038. doi: 10.1093/hmg/dds343

Figure 9.

Figure 9.

OCRL's 5-phosphatase activity regulates phosphorylation of cofilin. (A) HeLa cells were transfected with OCRL- or GFP-specific siRNA, stimulated with 10 ng/ml EGF for 20 min, fixed and analyzed by confocal laser scanning microscopy. Cells were stained with an anti-phospho-cofilin antibody, followed by an AlexaFluor488-conjugated secondary antibody (green). Scale bar = 10 µm. To exclude off-target effects, three different siRNAs against OCRL were independently applied. (B) Quantification of the mean fluorescence of cells stained for phospho-cofilin 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 is expressed as percentage of control cells. Results are from three independent experiments with 10 sections per experiment evaluated, and are shown as the mean ± SD. ***P < 0.001 (two-tailed Student's t-test). (C) HeLa cells were treated with siRNA against OCRL, followed by transfection with siRNA-resistant constructs expressing HA-tagged wild-type OCRL isoform a (HA-OCRL a) or the 5-phosphatase-inactive OCRL isoform a (HA-OCRLR493A/R500T a), prior to stimulation with 10 ng/ml EGF for 20 min, fixation and immunofluorescence analysis. OCRL variants were detected by staining cells with an anti-HA 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 an siRNA-resistant OCRL construct. Scale bar = 10 µm. (D) 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 HA-OCRL a or HA-OCRLR493A/R500T a is expressed as percentage of cells depleted of OCRL without OCRL re-expression. Results are from 10 different sections per condition, and are shown as the mean ± SD. ***P < 0.001 (two-tailed Student's t-test).