Caveolae generation down-regulates CLIC endocytosis.

<p>(A) Cell lysates from untransfected Cavin-1−/− MEFs or Cavin-1−/− MEFs transiently transfected with CAV1-YFP or Cavin-1-mCherry or co-transfected with CAV1-YFP and Cavin-1-mCherry were immunoblotted for CAV1 and Cavin-1. Actin was used as loading control. For quantification, the integrated intensity of protein bands was calculated. (B) Representative image of cell from (A) showing colocalization between CAV1 and Cavin-1 at different subcellular locations. Insets depict CAV1, Cavin-1 and merged labeling at the plasma membrane and cytosolic regions, marked by the square. 40 cells from (A) were subjected to Pearson correlation analysis, and a representative graph is shown. (C) Cells from (A), growing on coverslips, were subjected to internalization assay with anti-CD44 mAb and Tfn-647 for 2 min at 37°C. Cells were acid washed prior to fixation and internalized anti-CD44 mAb was labeled with AF-555 secondary antibody. 40–50 cells were quantified for normalized fluorescent intensity of endocytic markers. (D) FRAP analysis for CAV1 diffusion at the plasma membrane of Cavin-1−/− MEFs expressing CAV1-YFP alone or co-expressing CAV1-YFP and Cavin-1-mCherry. Fluorescence recovery curve and lateral diffusion analysis (inset) from 15 cells (mean ± SD) are shown. In (B) data represent mean ± SEM of data pooled from three independent experiments and in (C) data represent mean ± SEM of three independent experiments. ***p<0.0005,****p<0.0001 (two-tailed t-test). Scale bar: 10 µm.