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Fig. 9. TOM1-specific antibody can block the ability of the C-terminal region of TOM1 to bind clathrin. (A) Recombinant GST-TOM1(300-492) bound to glutathione-sepharose beads was pre-incubated with the indicated amounts of anti-TOM1 antibodies or rabbit IgG (as control). The beads were then washed and incubated with A431 cytosol in pull-down experiments. The input (10%) and pull-down materials were resolved by SDS-PAGE and analyzed by immunoblotting with either anti-clathrin heavy chain (upper panel) or anti-GST (lower panel) antibody. The amounts of TOM1 antibodies bound to the GST fusion beads were visualized by western blotting directly with anti-rabbit IgG conjugated to horseradish peroxidase (middle panel). (B) TOM1-specific antibody disrupts the cellular distribution of clathrin. A431 cells were microinjected with anti-TOM1 or rabbit IgG as a control. The cells were then fixed and permeabilized with 0.05% saponin before staining with FITC-conjugated anti-rabbit IgG to visualize the microinjected antibodies (left panels) and anti-clathrin heavy chain (right panels) to detect the distribution patterns of clathrin. Bar, 10 µm.
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