Cavin-1 regulates cholesterol distribution and Cdc42 activity.

<p>(A) Filipin labeling was performed for Cavin-1-GFP-expressing CAV1−/− MEFs, and for quantification average fluorescence intensity measured from 40–50 transfected cells was normalized to untransfected cells. (B) Whole cell lysates from CAV1−/− MEFs expressing CAV1-YFP and Cavin-1-GFP respectively were immunoblotted for Cdc42 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. The bar graph represents quantitation of protein levels, calculated by measuring band intensities by densitometry. (C) Pearson coefficient analysis determined at the plasma membrane for CAV1/Cavin-1 (positive control), Cavin-1/Cdc42, and randomized Cavin-1/Cdc42. (D) CAV1−/− MEFs were co-transfected with CRIB-YPet and Cavin-1-mCherry and quantitative colocalization analysis was performed at the PM ruffles. For 30 cells, line scans (10 pixels' length) were performed at random positions on PM ruffles and cytosol. The resultant fluorescent intensities profiles were plotted against each other and subjected to linear regression analysis. (E) CAV1−/− MEFs were transiently transfected with Cdc42-CyPet alone, with Cdc42-CyPet and CRIB-YPet simultaneously, or with Cdc42-CyPet, CRIB-YPet, and Cavin-1-Flag. Lifetime of CyPet was analyzed by FLIM-FRET and a representative graph showing lifetime of CyPet in 25 cells, mean ± SEM, is shown. (F) In Cavin-1−/− and CAV1−/− MEFs internalization assay was performed with anti-CD44 mAb and Tfn-647 for 2 and 10 min at 37°C. Endogenous CAV1 and Cavin-1 was labeled with respective primary antibodies followed by AF-488 secondary antibody labeling and for internalized anti-CD44 mAb labeling AF-555 secondary antibody was used. In (A,B,D) data represent mean ± SEM of three independent experiments. In (C) data represent average mean ± SEM of pooled data from three independent experiments; n = 40 cells. **p<0.01,***p<0.001 ****p<0.0001 (two-tailed t-test). Scale bar: 10 µm.