Fig. 1. Interaction between TOM1 and clathrin. (A) Identification of clathrin as a binding protein for TOM1. 300 µg recombinant GST or GST-TOM1(300-492) immobilized on glutathione-Sepharose beads, were incubated overnight with 85 mg pre-cleared rat brain cytosol at 4°C. Upon washing, proteins bound to the columns were eluted and resolved by SDS-PAGE and visualized by Coomassie Blue staining of the gel. Peptide sequences derived from the 220 kDa protein band were obtained by mass spectrometry: 1. AYEFAER; 2. TPDTIRR; 3. HELIEFR; 4. ENPYYDSR. (B) The C-terminal region of TOM1 interacts with clathrin. GST or GST-TOM1(300-492) immobilized on glutathione-Sepharose beads were incubated with A431 cytosol. The beads were washed and bound proteins as well as input (4%) were resolved by SDS-PAGE. 50 µg of input lysate and post-pull-down extracts were precipitated with trichloroacetic acid and analyzed by SDS-PAGE. Immunoblot analysis was performed with anti-clathrin heavy chain (HC) antibodies (upper and middle panels). GST and GST fusions were visualized by immunoblotting with anti-GST antibody as a control for levels of fusions used in pull-down experiments (bottom panel).