Figure 6.

BFA prevents complex glycosylation of β2, a fraction of which can reach the cell surface. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and then treated 2 h later with BFA (+) or left untreated (−) and grown overnight in wells. A and C, cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼100 μg), and the corresponding portion (nine-tenths) was subjected to overnight pulldown. Denatured protein from cell lysates and pulldowns was treated overnight at 37 °C with Endo H to cleave off immature N-glycans or left untreated (−). Representative Western blots show that the (lower) faster-migrating band of β2 WT is the only one visible in cells treated with BFA and increases its mobility with the Endo H treatment; this band coincides with unglycosylated β2 (C; compare with Fig. 2A). Note that Endo H digestion in pulldowns is only partial, either due to saturation of the enzyme or to suboptimal conditions for enzyme action. Blots for Na/K-ATPase are included as loading controls. Molecular mass markers are in kDa. B, band quantitation shows reduced levels of immature β2 in biotin-NeutrAvidin pulldowns (Membrane) of BFA-treated cells. Two-tailed Student's t test shows significant difference (*, p < 0.05). Data are mean ± S.D. (error bars) (n ≥ 3). D, cells were fixed and immunostained with a rabbit polyclonal antibody against calnexin (red) and a mouse monoclonal to GM130 (blue). Representative xy sections show that, in BFA-treated cells, β2 WT displays an intracellular accumulation comparable with mutated β2 (green), grossly overlapping with calnexin in enlarged structures (arrowheads). This contrasts with its apparent localization in the plasma membrane in untreated cells, displaying also a scattered pattern that does not overlap with calnexin (sections were taken at the cell level where β2 is mainly found in each case). Nuclear staining by DAPI is shown in gray. Scale bar, 10 μm.