Figure 9.

Effect of MAL depletion on the overall apical delivery of membrane proteins in MDCK and FRT cells. MDCK and MDCK/hMAL-ΔN cells (A and B) or FRT and FRT/hMAL-ΔN cells (C and D) were transfected with AM or AS oligonucleotide, plated on tissue culture inserts, and incubated at 37°C for 48 h. To study surface delivery of membrane proteins, cells were repeatedly treated with sulfo-SHPP from both the apical and basolateral faces of the insert. After incubation at 37°C for 7 h, the appearance of new surface proteins was monitored by domain-selective labeling with sulfo-NHS-biotin. After cell lysis, the extracts were subjected to SDS-PAGE and analyzed with streptavidin-peroxidase. Endogenous MAL and exogenous hMAL-ΔN were separately analyzed by immunoblotting with mAb 2E5 and 9E10 (MDCK cells), respectively, or simultaneously with mAb 6D9 (FRT cells). (A and C) Representative experiment. (B and D) Quantitative analysis of the effect of MAL depletion on the polarized delivery of membrane proteins. The intensity of the apical and basolateral signal corresponding to newly delivered surface proteins from experiments in which MAL was depleted at different extensions was quantified by densitometric analysis of the entire lanes. The values obtained are expressed as percentages of the apical (black bars) or (gray bars) basolateral delivery obtained in normal cells transfected with the control AM oligonucleotide. The results of some representative experiments are shown. The effects observed in other experiments in which MAL was depleted to different extents (our unpublished results) were consistent with those shown.