FIGURE 5.

FLAG-S1R binds to GST-ELMODs and inhibits the ARL2 GAP activity of GST-ELMOD1 or GST-ELMOD2. A, reciprocal co-immunoprecipitations of GST-ELMOD and FLAG-S1R reveals specific binding of S1R to all three ELMODs. All proteins were tagged (FLAG-S1R or GST all others) and co-expressed in HEK293T cells (as indicated along the top), and used in reciprocal pulldown assays using glutathione-Sepharose or anti-FLAG antibodies (as indicated on the left side of each panel). Each panel shows an immunoblot, using the antibody indicated at the right. WL, whole cell lysate; PD, pulldown. The key panels are the top panel, which shows GST pulldowns from each ELMOD expressed also enriches for FLAG-S1R, and the third panel down, which shows FLAG pulldowns each of the GST-ELMODs. GST and GST-ARL13B are used as negative controls for the ELMOD proteins; note the lack of interactions with FLAG-S1R. The empty FLAG vector is indicated as FLAG at the top but because of the small size of the recombinant protein does not appear in any of the immunoblots. A nonspecific band is seen in all lanes using the FLAG antibody, running right below the FLAG-S1R. B, ARL2 GAP assays for GST-ELMOD1 or GST-ELMOD1-S1R are shown as a function of increasing GST-ELMOD1 concentrations. Equal amounts of GST-ELMOD1, alone or complexed with S1R, were assayed. C, the same as panel B, except GST-ELMOD2 is used instead of GST-ELMOD1.