FIG. 4.

BAF binds directly to MA with low micromolar affinity. (A) BAF binds directly to MA, but not CA, in blot overlay assays. Different volumes of protein lysate from induced (+ IPTG) or uninduced bacteria (− IPTG) containing plasmids encoding either MA or CA were resolved by SDS-PAGE. Gels were either transferred to filters and probed with ³⁵S-labeled BAF or stained with Coomassie blue to verify equal protein loads. (B) MA binds BAF in coimmunoprecipitation assays. Purified recombinant MA (200 ng) was incubated with (+) or without (−) 200 ng of recombinant BAF and then immunoprecipitated using protein A beads with or without antibodies against MA. A fraction (15%) of each pellet (P) and 30% of each supernatant (S) were resolved by SDS-PAGE and immunoblotted for BAF and MA, as indicated with antibodies to BAF (α-BAF) and MA (α-MA). (C) The equilibrium affinity of MA for BAF was determined in microtiter well assays by adding increasing concentrations of ³⁵S-labeled MA to constant amounts of recombinant BAF immobilized in microtiter wells. Double-reciprocal plots (not shown) were used to determine the affinity constant as described previously (30).