Fig. 4.

AAGAB is required for both AP1- and AP2-mediated cargo trafficking. (A) Representative immunoblots from three experiments showing the expression of the indicated proteins in WT and mutant HeLa cells. M.W., molecular weight. (B) Normalized surface levels of TfR in WT and mutant HeLa cells measured by flow cytometry. Cells were disassociated by Accutase and stained with monoclonal anti-TfR antibodies and APC-conjugated secondary antibodies. APC fluorescence measurements of ∼5000 cells were collected on a CyAn ADP analyzer. Mean APC fluorescence of mutant cells was normalized to that of WT cells. Data are presented as mean±s.d., n=3. ***P<0.001. P values were calculated using one-way ANOVA and Dunnett's multiple comparison test. (C) Representative confocal microscopy images from three experiments showing surface levels of TfR in WT and mutant HeLa cells. Surface TfR levels of non-permeabilized cells were labeled using anti-TfR antibodies and Alexa Fluor 568-conjugated secondary antibodies (red). Nuclei were stained with Hoechst 33342 (blue). Images were captured using a 100× oil immersion objective on a Nikon A1 Laser Scanning confocal microscope. Scale bars: 20 µm. DKO, double knockout.