FIGURE 8.

Susceptibility of mature and immature forms of cysless-WT and D164C/D805C mutant P-gp to digestion by PNGase F and Endo H. A, Western blot analysis of total cell lysates of HeLa cells transduced with cysless-WT or D164C/D805C mutant P-gp when grown in the absence or presence of 6.25 μm CsA or CFTR correctors C3 and C4 (20 μm). Cell lysate proteins (3 μg) were loaded in each lane, and immunoblotting with C219 antibody was performed as described under “Experimental Procedures.” Lane 1, cysless-WT; lane 2, cysless-WT treated with 6.25 μm CsA; lane 3, cysless-WT treated with 20 μm corrector C3, lane 4, cysless-WT treated with 20 μm corrector C4; lane 5, D164C/D805C; lane 6, D164C/D805C treated with 6.25 μm CsA; lane 7, D164C/D805C treated with 20 μm C3; lane 8, D164C/D805C treated with 20 μm corrector C4. Levels of β-actin are shown as loading controls for each lane in the immunoblot. Arrows show the position of mature P-gp and β-actin, whereas an arrowhead marks the position of the immature P-gp. B, glycosidase digestion of cell lysates (2 μg of protein) from cysless-WT and double mutant P-gp was loaded in each lane. Cell lysates from HeLa cells transduced with cysless-WT (lanes 1, 2, and 3) and D164C/D805C (lanes 4, 5, and 6) and grown in the absence of CsA were subjected to Endo H or PNGase F digestion as described under “Experimental Procedures” and processed for immunoblotting using C219 antibody. The complex glycosylated (Band A, mature protein) and deglycosylated (Band B, immature protein) forms are indicated. C, glycosidase digestion of crude membranes from High-Five insect cells (0.5 μg of protein) expressing cysless-WT (lanes 1, 2, and 3) and D164C/D805C (lanes 4, 5, and 6) P-gp. In both cases the digest was resolved on 7% Tris-acetate gel and subjected to Western blotting using C219 antibody. The details of treatments are given in the figure, and blots from a representative experiment are shown. Similar results were obtained from three independent experiments.