Pitstop 2 Is a Potent Inhibitor of Clathrin-Independent Endocytosis (original) (raw)
Figure 1
Pitstop 2 inhibits both clathrin-dependent and clathrin-independent endocytosis.
(A) Hela cells were preincubated with DMSO (control) or 20 µM pitstop 2 or pitstop 2-negative control for 15 min. Cells were allowed to internalize Alexa594-Transferrin and antibodies to MHCI for 30 min at 37°C in the presence of DMSO or drugs. Surface bound antibodies were removed by low pH wash to visualize the internalized proteins (Internal) prior to fixation or cells were fixed immediately after the internalization to label for the internal and surface pools (Total) of the protein. Cells were then labeled with secondary antibodies to detect MHCI. (B) Cells preincubated with DMSO or 20 µM pitstop 2 were allowed to internalize antibodies directed towards CD59, CD44, CD98 and CD147 for 30 min and fixed. Surface bound antibodies were removed by acidic wash or blocked with unlabeled goat-anti-mouse IgG. Cells were then incubated with secondary antibodies to detect the internalized cargo proteins. The results shown are representative of three independent experiments. Bar, 10 µm.