Treatment with mitomycin C (MMC) increases Pgp-EGFP fusion protein at the cell surface.

(A) hCMEC/D3-MDR1-EGFP (doxycycline-on) cells were treated with MMC (1 µM) for 2 or 4 h and analyzed at the end of the 2 h-exposure period or 20 h after the 4 h-exposure period. Western blot analyses of cell surface proteins isolated via EZ-Link Sulfo-NHS-SS-Biotin-Neutravidin assay revealed an obvious increase of Pgp-EGFP abundance at the cell surface after MMC exposure, whereas no effect on intracellular Pgp was seen. One representative result of six independent experiments is shown. In (B), Pgp-EGFP bands of the cell surface were analyzed densitometrically and Pgp signals were normalized relative to the coomassie-stained portion of the gel. Data variability is shown as ± SEM of six experiments; significant differences of treated vs. untreated samples are indicated by asterisk (P<0.05). (C, D) No significant induction of Pgp-EGFP fusion protein was measured in whole cell lysates at the same exposure conditions used in (A). Respective P values in D were 0.5502 (2 h MMC) and 0.2534 (24 h MMC). Pgp-EGFP bands were analyzed densitometrically and Pgp signals were normalized on actin. Data variability is shown as ± SEM of three experiments.