Figure 7.

MAL depletion affects the polarized transport of p75NTR in FRT cells. (A) FRT cells or FRT/hMAL-ΔN cells were transfected with AM or AS oligonucleotide, plated on tissue culture inserts, and incubated at 37°C for 48 h. Cells were then infected with recombinant adenovirus expressing p75NTR, and 8 h later the cell surface was labeled with sulfo-NHS-biotin from either the apical or the basolateral faces of the insert. After cell lysis, the extracts were immunoprecipitated with anti-p75NTR antibodies, and p75NTR was detected by immunoblotting with streptavidin-peroxidase. The endogenous (rMAL) or exogenous (hMAL-ΔN) MAL levels in these cells were examined by immunoblot analysis with mAb 6D9. The intensity of the apical and basolateral signal corresponding to surface p75NTR was quantified. The values obtained are represented as percentages of surface p75NTR on the apical (black bars) or basolateral (gray bars) subdomain. (B) FRT and FRT/hMAL-ΔN cells 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 endogenous DPPIV, 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 DPPIV molecules was monitored by domain-selective labeling with sulfo-NHS-biotin. After cell lysis, the extracts were immunoprecipitated with anti-DPPIV antibodies and were subjected to SDS-PAGE and analyzed with streptavidin-peroxidase. Endogenous (rMAL) and exogenous (hMAL-ΔN) MAL levels were analyzed by immunoblotting with mAb 6D9. The intensity of the apical and basolateral signal corresponding to newly delivered surface DPPIV was quantified. The values obtained are represented as percentages of the DPPIV delivered to the apical surface in MAL-depleted cells relative to that in control cells during the 7-h period of chase. No basolateral signal was detectable for DPPIV under any of the conditions used.