Cavin-1 and Cavin-3, but not Cavin-2 and Cavin-4 negatively regulate CLIC endocytosis.

<p>(A) CAV1−/− MEFs were transiently transfected with pIRES-GFP, pIRES-Cavin-1, pIRES-Cavin-2, pIRES-Cavin-3, and pIRES-Cavin-4 respectively and internalization assay was performed 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. (B) 40–50 cells from each transfection were quantified for normalized fluorescent intensity of endocytic markers. (C,D) 3T3-L1 cells were transiently transfected with siRNA directed specifically to Cavin-1 and Cavin-3 respectively. Uptake and quantification of internalized marker were performed as mentioned in (A,B). In (B–D) data represent mean ± SEM of three independent experiments. *p<0.05,**p<0.005,****p<0.0001 (two-tailed t-test). Scale bar: 10 µm.