Fig. 12. The 9-kDa protein interacts with VAMP7, apoB48 and L-FABP in a phosphorylation-dependent manner. (A) Intestinal ER (500 µg) was incubated for 30 minutes at 37°C with native intestinal cytosol (1 mg) and an ATP-regenerating system. The reaction was stopped by placing the incubation tube on ice and adding cold HEPES buffer (10 mM, pH 7.2). After incubation, the ER was isolated, solubilized in cold PBS containing 2% Triton X-100 and incubated with 20 µl primary antibodies (anti-rabbit IgG, anti-VAMP7, anti-apoB48 or anti-L-FABP) for 2 hours at 4°C. Appropriate secondary antibodies bound to agarose beads were added and allowed to incubate overnight at 4°C. Immune complexes bound to the beads were isolated, washed ten times, and proteins separated on 8%-16% SDS-PAGE. The gel was stained with SimplyBlue^TM SafeStain (Invitrogen) to identify the proteins. Only the low-molecular-weight bands are shown. (B) A reaction similar to that shown in A was performed, with the exception that PKC-immunodepleted intestinal cytosol and 2 M urea-washed intestinal ER was used with an ATP-regenerating system. Similar to A, the reaction was stopped post incubation, the ER isolated and solubilized ER membranes were subjected to immunoprecipitation using 20 µl primary antibodies (anti-rabbit IgG, anti-VAMP7, anti-apoB48 or anti-L-FABP). The bead-bound immune complexes were isolated and proteins separated on 8%-16% SDS-PAGE. The proteins were identified by staining with SimplyBlue SafeStain. Only the low-molecular-weight bands are shown. The primary antibodies used are shown above each lane.