FIG. 3.

BAF binds directly to p55 Gag. (A) Blot overlay assay. Purified p55 protein (125, 250, and 500 ng), RT (250, 500, and 1,000 ng) and IN (250, 500, and 1,000 ng) proteins (prot.) were resolved by SDS-PAGE, transferred to filters, and probed with ³⁵S-labeled BAF. The autoradiograph is shown. (B) In vitro coimmunoprecipitation assay. Recombinant p55 Gag protein (500 ng) was incubated with (+) or without (−) 200 ng of recombinant BAF and then immunoprecipitated using protein A beads with (+) or without (−) antibodies against BAF (BAF Ab). One-tenth of each pellet (P) and 10% of each corresponding supernatant (S) fraction were resolved by SDS-PAGE and immunoblotted with antibodies specific for either BAF or the MA domain of p55 Gag. (C) Binding affinity. The affinity of BAF for p55 Gag was determined in microtiter well assays. Increasing concentrations of ³⁵S-labeled BAF were incubated with constant amounts of recombinant p55 Gag (1.8 pmol) immobilized in microtiter wells. Double-reciprocal plots (not shown) were used to determine the affinity constant as described previously (30). (D) Endogenous BAF coimmunoprecipitates with p55 Gag in human (HeLa) cell extract. Full-length Gag (pCiGagPRE) was expressed in HeLa cells, and protein lysates (L) from transfected cells were incubated with protein A beads alone (Protein A) or protein A beads plus antibodies against MA (α-MA Ab). A fraction (15%) of the supernatant (S) and 30% of each immunoprecipitate (P) were resolved by SDS-PAGE and immunoblotted for BAF. Transf., transfected.