Figure 2.

Surface MHCI and Tac are internalized independently of dynamin, are not in rafts, and do not colocalize with clathrin at the PM. (A) HeLa cells were transfected with either Tac (squares) or Tac-LL (circles) and either the wild-type (open symbols) or K44A mutant (filled symbols) of dynamin-2. Tac internalization was monitored by Cell ELISA using biotinylated anti-Tac antibody as described in MATERIALS AND METHODS, and the fraction of Tac antibody remaining on the surface at different time points was recorded. A representative experiment is shown with the mean and SD for triplicates of each time point. (B) HeLa cells were transfected with either Tac, Tac-LL, or Tac-GPI, treated with 1.0% Triton X-100 at 4°C for 3 min, and then fixed. Surface Tac proteins were assessed by incubation with anti-Tac antibodies (without saponin). (C) HeLa cells were transiently transfected with Tac or Tac-LL and fixed. Surface labeling was performed using rabbit anti-Tac antibody in the absence of saponin, followed by a second fixation. Cells were then labeled in the presence of saponin with mouse anticlathrin antibody and with secondary antibodies. Background, surface fluorescence staining was diminished in these images to enhance the punctate pattern of the proteins. Magnified insets are designated. Bar, 10 μm.