FIG. 4.

Sec13 and Nup93 interact physically with MAD. (A) Sec13 preferentially interacts with phosphorylated MAD. The indicated expression vectors were transfected into S2 cells, together with Punt/Tkv to phosphorylate MAD. The whole-cell extracts (WCE) were immunoprecipitated (IP) with anti-FLAG, and the bound proteins were analyzed by immunoblotting (IB) with the indicated antibodies. The asterisk marks a protein comigrating with Sec13 that cross-reacts with the anti-FLAG antibody. (B) S2 cells transfected with HA-Sec13 only were treated with Dpp, and the cell lysate was subjected to immunoprecipitation with anti-HA. The bound proteins were examined by IB using anti-MAD. The arrow points to endogenous MAD. The specificity of the anti-MAD antibody has been demonstrated (data not shown). (C) Cell extracts from S2 cells transfected with FLAG-MAD and Punt/Tkv only were immunoprecipitated with anti-FLAG, and the bound proteins were examined by IB with anti-Sec13. (D) Experiments similar to those described for panel A were carried out to examine the Nup93-MAD interaction. (E and F) GST pulldown assays to test direct protein interactions using purified recombinant FLAG-MAD (after phosphorylation by Punt/Tkv) and GST-Sec13 or GST-Nup93, with GST serving as the control. The bound proteins were analyzed by IB with anti-FLAG. (G and H) S2 cells were treated with the indicated RNAi and were transfected with the indicated plasmids. The cell extracts were subjected to IP with anti-FLAG in order to test interactions between MAD and Sec13 or Nup93. Quantitative real-time PCR confirmed that Nup75 knockdown was >70%. The asterisks indicate a band cross-reacting with anti-FLAG.