Figure 4

In vitro binding interactions between MoMLV and HIV-1 integrases and selected proteins identified in the yeast two-hybrid screen. In vitro binding assays between the pmalc2 empty vector (MBP), full-length pmalc2-MoMLV IN (mIN) or full-length pmalc2-HIV-1 IN (hIN) and seventeen of the clones isolated in the screen, plus mLEDGF expressed as GST fusions. The MBP fusion lysates were incubated with amylose resin, washed extensively, resuspended in equal volumes of buffer, and then aliquoted to separate tubes. These tubes were incubated with the GST fusion lysates, washed and eluted with 15 mM maltose. 25 μl of each eluate was electrophoresed on 10 or 12% SDS-PSGE gels, transferred to PVDF membranes, and the same Western was probed with anti-GST, stripped, and then probed with anti-MBP. All Westerns are loaded from left to right: MBP, mIN, and hIN fusion reactions. All upper panels, anti-MBP. All lower panels, anti-GST. (A) Maltose binding protein fusions with empty GST vector; MBP fusions with Brd2, AF9, and Ankrd49. (B) MBP fusions with mLEDGF, Fen-1, Enx-1, and TFIIE-β. (C) MBP fusions with Ku70, PRC, Baz2b, and ABT1. (D) MBP fusions with SF3a3, U5snRNP, KIF3A, and Radixin. (E) MBP fusions with Znfp38, U2AF²⁶, and Ran bp10.