Fig. 3.

BCL11A interacts directly with SIRT1 in vitro. (A) In vitro translated and [³⁵S]Met-labeled full-length BCL11A and truncation BCL11A mutants were incubated with equivalent amounts of bacterially expressed GST (lane 2) or GST-SIRT1 fusion protein (lane 3). After extensive washing, [presup35S]Met-labeled BCL11A associated with the affinity resin was determined by SDS–PAGE and autoradiography. Input [³⁵S]Met-labeled proteins are shown in lane 1. BCL11A truncation mutants used in these studies (B–F) are schematically represented on the right with zinc finger motifs denoted by vertical bars. (B) A schematic representation of full-length SIRT1 and SIRT1 truncation mutants used to generate GST-SIRT1 fusion proteins for in vitro pulldown experiments (see below). (C) [³⁵S]Met-labeled BCL111A 194–378 (the minimal SIRT1-interaction domain) was incubated with equivalent amounts of GST (lane 2) or GST-SIRT1 fusion proteins (lanes 3–8). The position of bound [³⁵S]Met-labeled BCL11A 194–378 is indicated by an arrow on the left. Lane 1 corresponds to 10% of the [³⁵S]Met-labeled BCL11A 194–378 that was incubated with GST or GST-SIRT1 fusion proteins. Shown in (A and C) are representative experiments that were replicated three to five times.