Figure 11.

Wild-type Rac1 counteracts accumulation of MPR300 in enlarged endosomal vesicles. (A) HeLa cells were treated with OCRL-specific siRNA, followed by transfection with constructs expressing myc-tagged Rac-wt or RacN17, prior to fixation and immunofluorescence analysis. Rac1 variants were detected by staining cells with an anti-myc antibody, followed by an AlexaFluor546-conjugated secondary antibody (red). MPR300 localization was visualized by using an anti-MPR300 antibody, followed by an AlexaFluor488-conjugated secondary antibody (green). * indicates cells transfected with a Rac1 expression vector. The nucleus was visualized using DAPI (blue). Scale bar = 10 µm. (B) Quantification of MPR300-positive vesicles of various sizes was performed by the Metamorph software. To enable direct comparison, all images were taken under the same magnification and laser intensity settings. Sizes of MPR300-positive structures are given in arbitrary units (AU). The number of MPR300-positive vesicles per AU (≤50, 51–100, 101–150 and ≥151) is given as percentage of the number of vesicles counted in total. Results are from three independent experiments with 10 sections per condition of each experiment evaluated, and are shown as the mean ± SD (black square OCRL siRNA, white square OCRL siRNA+Rac-wt, gray square OCRL siRNA+RacN17). **P = 0.008; ***P < 0.001 (two-tailed Student's t-test).