FIGURE 2.

RIBEYE(B) specifically interacts with Munc119 in fusion protein pull-down assays. Pull-down analyses of RIBEYE(B)/Munc119 complexes using bacterially expressed fusion proteins. In A, pull-down experiments were analyzed by Coomassie Blue-stained polyacrylamide gel after SDS-PAGE. In B, by Western blot analyses with the indicated antibodies. A and B, lanes 1–4 show the indicated purified fusion proteins (input fractions). All input lanes, except for lane 4, represent 50% of the input fraction. Lane 4 represents 25% of the input fraction. In lanes 5–8, 100% was loaded. GST-tagged fusion proteins were used as immobilized bait proteins and MBP-tagged proteins as soluble prey proteins. Only Munc119-GST pulled-down RE(B)-MBP (lane 8) but not GST alone (lane 6). Neither GST alone nor Munc119-GST pulled-down MBP alone (lanes 5 and 7). The asterisks in lanes 3, 7, and 8 of A label a break-down product of Munc119-GST. SDS-PAGE clearly demonstrated that Munc119-GST does not pull-down MBP alone (A). To further exclude that any MBP is nonspecifically pulled-down by Munc119-GST, we also analyzed the results of the pull-down assays by Western blotting with anti-MBP antibodies. B, Western blot analyses with anti-MBP antibodies (B) clearly show that only RE(B)-MBP (lane 8) but not MBP alone (lane 7) is pulled-down by Munc119-GST. GST alone does not pull-down RE(B)-MBP as well as MBP alone as shown by Western blotting with antibodies against MBP (Ba) demonstrating the specificity of the interaction and completely confirming the results in A. In Bb, the same blot as analyzed in Ba was reprobed (after stripping) with antibodies against GST to show equal loading of the bait proteins. Abbreviations: CB, Coomassie Blue.