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. 2010 Jun 14;30(16):4022–4034. doi: 10.1128/MCB.00124-10

FIG. 5.

FIG. 5.

Physical interaction between Msk and Sec13 or Nup93. (A) S2 cells were transfected with FLAG-Sec13, and the lysate was used for immunoprecipitation (IP) with anti-FLAG. The endogenous Msk was detected by immunoblotting (IB). (B) Sec13 overexpression did not significantly affect the Msk-MAD interaction. S2 cells were transfected with the indicated expression vectors. The whole-cell extracts (WCE) were immunoprecipitated with anti-FLAG, and the bound proteins were analyzed by IB as indicated. (C) FLAG-Nup93 was expressed in S2 cells, and anti-FLAG immunoprecipitation was used to detect its interaction with endogenous Msk. (D) GST pulldown experiment testing direct interactions between purified recombinant Msk and GST-Sec13 or GST-Nup93, with GST as the control. Proteins bound to GST beads were analyzed by IB with anti-Msk. (E) S2 cells were subject to the indicated RNAi and were then transfected with the indicated expression vectors. IP was carried out to test for interactions between Msk-V5 and FLAG-Sec13 or FLAG-Nup93. RNAi knockdown of Nup75 resulted in decreased Sec13-Msk and Nup93-Msk interactions. Quantitative real-time PCR confirmed that Nup75 knockdown was >70%. The asterisk indicates a band cross-reacting with anti-FLAG.