Immunofluorescence detection of cell surface recycled EGFR with prebound EGFR-ECD antibody

wt-PKCδ-myc or YF-PKCδ-myc BT-549 cells were grown in the presence of dox (1.0 µg/ml) for 2–3 d. Cells were then starved in low-serum medium (RPMI + 0.1% FBS) for 20 h in the presence of dox. For the assay, cells were prelabeled with mouse anti-EGFR-ECD antibody in the presence of 20 ng/ml EGF for 60 min at 4°C, after which medium was replaced with warm low-serum medium, and the cells were transferred to 37°C for endocytosis to occur for 15 min. After the pulse of endocytosis, cells were placed on ice and acid washed in 0.2 M acetic acid and 0.5 M NaCl, pH 2.5, for 5 min to remove Ab bound to EGFR remaining on the cell surface, followed by four washes in low-serum medium. Cells were then incubated at 37°C for 30 min in low-serum medium without EGF to allow recycling of EGFR back to the cell surface. At the end of the recycling period, the cells were placed on ice and fixed with 3.7% formaldehyde. Cells were washed twice in TBS, blocked in TBS/2% BSA, incubated with anti-mouse–Alexa Fluor 594–conjugated secondary Ab to detect EGFR-ECD Ab-bound EGFR that had returned to the cell surface, and then mounted using ProlongGold with DAPI. To determine the pool of EGFR internalized after the 15-min endocytosis pulse, an additional well was subjected to the pulse of endocytosis and then fixed, permeabilized, blocked, and stained with anti-mouse–Alexa Fluor 594–conjugated secondary Ab. For each time point, a minimum of six fields of view were quantified. Mean fluorescence intensity was quantitated using the analyze particles function in ImageJ.