VDU1 and VDU2 are ER-resident integral membrane proteins and colocalize with D2. (a) Subcellular fractionation of cell lysates transiently expressing GFP-VDU1, GFP-VDU2, or D10. Fractions of total cell lysate, particulate fraction, and cytosol were isolated and analyzed by Western blot using anti-GFP antibody. This experiment was performed twice. (b) Colocalization of VDU1 (top panels) and VDU2 (bottom panels) with D2. Cells transiently expressing the indicated pairs of proteins were permeabilized with Triton X-100 and processed for immunocytochemistry with anti-FLAG antibody. Visualization was achieved with goat anti-mouse Texas red F(ab′)₂ fragment. D2 is shown in red, while the VDUs are shown in green. The yellow bar indicates 10 μm. The distribution spectrum of image pixels is shown in the insets. (c) D2 activity in HEK-293 cells transiently coexpressing D2 and/or VDU1 or VDU2. Cells were harvested 48 hours after transfection. *P < 0.05 vs. D10-transfected cells. (d) D2 activity, as in c except that cells were treated with 0.2% DMSO (vehicle) or 100 μM cycloheximide (CX) and harvested 2 hours later. *P < 0.05 vs. DMSO-treated cells. Values in c and d are the mean ± SD of two to four cell plates; experiments were performed two to three times.