Figure 4.

Antagonism between Crm1 and Mae for binding to Yan is modulated by Yan phosphorylation. (A) Crm1 and Mae compete for Yan binding. Flag-tagged Yan purified from S2 cells was immobilized on anti-Flag agarose beads, and S2 cell nuclear extracts were incubated with the beads in the absence (lane 2) or presence of bacterially expressed His-tagged MBP-MaeSAM (lane 3) or MBP-MaeSAM^A141D (lane 4). (Lane 1) As a control, Yan was omitted from the binding reaction. Coprecipitated endogenous Crm1 was revealed by anti-Crm1 immunoblotting (upper panel), while coprecipitated Mae was revealed by anti-His immunoblotting (lower panel). (B) Immobilized Flag-Yan that had been expressed in S2 cells in the presence (lanes 1-6) or absence (lanes 7-12) of Ras^V12 was incubated with Crm1-containing nuclear extract and variable amounts of bacterially expressed MBP-MaeSAM. Coprecipitated endogenous Crm1 was revealed by anti-Crm1 immunoblotting (upper panel), while coprecipitated Mae was revealed by anti-His immunoblotting (lower panel). (C) Immobilized Flag-Yan^S127A that had been expressed in S2 cells in the presence (lane 3) or absence (lane 4) of Ras^V12 was incubated with Crm1-containing nuclear extract. Also shown are 15% of the input Crm1 (lane 1) and a negative control in which Yan was omitted from the binding reaction (lane 2). Coprecipitated endogenous Crm1 was revealed by anti-Crm1 immunoblotting (upper panel), while precipitated recombinant Yan was revealed by anti-Flag immunoblotting (lower panel).