For the IF assay, live PC9 cells were cultured on coverslips and incubated with mAbs (10 μg/ml) for 1 hour on ice before being washed three times with PBS. Cells were then cultured at 37°C for 0, 2, or 4 hours before fixation with 4% PFA. Antibodies were then labeled with Alexa Fluor 488–conjugated anti-mouse antibody. Images were acquired by Nikon confocal system A1Si.

For the FACS assay, live PC9 cells were incubated with mAbs (10 μg/ml) for 0.5 hour on ice before being washed three times with PBS. Cells were then cultured at 37°C for up to 4 hours before fixation with 4% PFA. Cells were then stained with Alexa Fluor 488–conjugated anti-mouse antibody and analyzed with FACS. Surface mean fluorescence intensity (MFI) was calculated. Surface mean fluorescent intensity (MFI), which represented surface localization of mAbs, was measured by FACS.

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