Fig. 8

Fig. 8

Membrane topology of zFxyd11a. (A) Homogenates of 293T cells expressing zFxyd11a-FLAG were fractionated into the cytosolic (lane 9) and membranous fractions. The membranes were incubated with PBS (lanes 1 and 2), 1 M NaCl (lanes 3 and 4), 0.1 M Na₂CO₃ (pH 11.5, lanes 5 and 6), or 1% Triton X-100 (lanes 7 and 8) followed by ultracentrifugation to separate soluble protein supernatants (S; lanes 1, 3, 5, and 7) from membranous pellets (P; lanes 2, 4, 6, and 8). The samples were analyzed by Western blotting with antibodies against FLAG, VTI1a (a TM protein), and DRP1 (a cytosolic protein). (B) HeLa cells were transfected with either zFxyd11a-FLAG (top panels) or FLAG-MHC-I (bottom panels) and were then processed for immunofluorescence staining with anti-FLAG antibody (red) without (left panels) or with (right panels) membrane permeabilization. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 20 μm. (C) HeLa cells transfected with zFxyd11a-FLAG were lysed directly (Control) or were subjected to cell-surface biotinylation (Biotinylated). Whole cell lysates (left panels) and biotinylated samples (right panels) were analyzed by Western blotting with anti-FLAG antibody (top panels) and anti-DRP1 antibody (bottom panels).