Amino acids 200–260 of SH2B1β is the site for FLNa interaction. A, Different in vitro translated myc-tagged truncated SH2B1β mutants were incubated with either GST (lanes 1, 3, and 5) or GST-tagged FLNa-4 (lanes 2, 4, and 6). Bound myc-SH2Bβ mutant was detected by IB with αmyc. Lanes 1 and 2, Amino acids 1-105 of SH2B1β; lanes 3 and 4, amino acids 1-200 of SH2B1β; and lanes 5 and 6, amino acids 1-260 of SH2B1β. B, mRFP-tagged (200–260) amino acids mutant of SH2B1β was translated in vitro in the presence of 35S methionine and incubated with either GST (lane 1) or GST-tagged FLNa-4 (lane 2). Bound mRFP-SH2Bβ mutant was detected by autoradiogram (48 h of film exposure). Equal amounts of 35S methionine-labeled SH2B1β input is shown on the right (24 h of film exposure). Amount of GST or GST-FLNa-4 was detected by IB with αGST. Each experiment was performed at least three times with similar results.