Robust trafficking of EGFR to the PM upon transferring expressing cells to 37°C following 14 hrs at 22°C. Live cells transiently expressing EGFR-GFP were harvested for flow cytometry, then fixed at each time point. Cells were incubated with an anti-N-terminal EGFR antibody followed by Alexa647-anti-IgG to label PM-associated EGFR, then imaged by confocal microscopy (A) or analyzed by flow cytometry (B) to determine the ratio of PM-localized EGFR fluorescence to total-EGFR fluorescence. Plot is representative of typical results; approximately 8,000 EGFR-GFP expressing cells were analyzed at each time point, and the ratios for all time points were normalized to that at 8 hr. Scale bars in A are 5 μm. (C) Representative flow cytometric scatter plots. RBL-2H3 cells transiently expressing GFP-tagged EGFR constructs were harvested, fixed, and labeled with anti-N-terminal EGFR antibody followed by Alexa 647-anti-IgG. Cells were gated on positive GFP fluorescence (blue and purple populations), and Alexa 647 fluorescence was analyzed to determine PM localization (purple population). Bold numbers indicate the mean Alexa 647 fluorescence of the GFP-positive population. Plots are representative of data used to generate “control” traces in Figures 1, 2, 4, and 7.