Pitstop 2 Is a Potent Inhibitor of Clathrin-Independent Endocytosis (original) (raw)
Figure 5
Pitstop 2 inhibition is not dependent upon AP2 or clathrin.
Hela cells were treated with control siRNA (A) or siRNA to deplete cells of μ2 subunit of AP2 (B) or clathrin heavy chains (C) as described in Materials and Methods. (A–C) Cells were preincubated with DMSO or 20 µM pitstop 2 for 15 min. Alexa488-Tfn and antibodies to MHCI were allowed to internalize for 30 min and processed as in Figure 1. Bars, 10 µm. (D) Detergent lysates of Hela cells treated with control siRNA, μ2 siRNA or CHC siRNA were separated by SDS-PAGE and immunoblotted with antibodies against proteins indicated at the left. The gel shows the level of knock-down from one representative experiment. The means and standard deviation of three separate experiments are plotted in the lower panel showing levels to be 14% and 12% of control levels for μ2 and clathrin heavy chain, respectively.