FIGURE 5.

Biotinylation of bEnd.3 cell surface proteins. A, representative Western blot of whole cell lysates of bEnd.3 cells in the absence or presence of either 5 mm KCN or 8 μg/ml of FCCP for 10 min. Total protein (20 μg) was loaded into each lane and probed with GLUT1 C-terminal antibody. B, quantitation of Western blot band density. Ordinate, relative expression (%); abscissa, experimental condition −PBS, PBS + KCN, and PBS + FCCP. C and E, representative Western blots of cell surface biotinylated bEnd.3 cell proteins obtained in the absence and presence of either 5 mm KCN or 8 μg/ml FCCP. Cells were poisoned at 37 °C for 10 min before cooling to 4 °C and biotinylation. 30 μg of total streptavidin-pulldown protein was loaded onto each gel and blotted with either GLUT1 C-terminal antibody (C) or antibody raised against Na⁺,K⁺-ATPase (E). Band densities were quantitated and shown in D (GLUT1) and F (Na⁺,K⁺-ATPase). Ordinate and abscissa are as in B. D, results are shown for total C-terminal antibody-reactive species (open bars), for 55-kDa C-terminal antibody-reactive species (gray bars), and for 48-kDa C-terminal antibody-reactive species (black bars). Each experiment was repeated at least three times, and the results of quantitations (B, D, and F) are shown as mean ± S.E.