FIG 1.

SH2B1β interacts with STAT3. (A) Domains of SH2B1β and SH2B1β deletion mutants. SH2B1β contains three proline-rich domains (P), a dimerization domain (DD), a nuclear localization signal (NLS), a nuclear export sequence (NES), a PH domain, and an SH2 domain. SH2B1β(R555E) is a dominant-negative mutant with a point mutation of arginine to glutamic acid at residue 555. (B) COS7 cells were transiently transfected with GFP, GFP-SH2B1β(1-670), GFP-SH2B1β(R555E), GFP-SH2B1β(1-260), GFP-SH2B1β(270-670), and GFP-SH2B1β(390-670) (left). Cell lysates were immunoprecipitated using anti-GFP antibody and resolved via SDS-PAGE followed by immunoblotting with anti-GFP and anti-STAT3 antibodies. Arrowheads indicate the overexpressed proteins. Cell lysates from COS7 cells expressing myc-SH2B1β were immunoprecipitated using anti-IgG or anti-myc antibody and resolved with SDS-PAGE, followed by immunoblotting using antibodies against STAT3, STAT1, STAT5b, and SH2B1. (C) COS7 cells were transiently cotransfected with vector only or myc-SH2B1β along with plasmid containing STAT3- or STAT-binding sequences fused to firefly luciferase (pLucTKS3 or pm67⁴) and pEGFP. Cells were harvested, and the firefly luciferase activity was measured and normalized to pEGFP levels. Values are the means ± standard errors of the means (SEM) from three independent experiments. (D) COS7 cells were transiently transfected with GFP-SH2B1β(ΔNES) or GFP-SH2B1β(ΔNLS). Cell lysates were fractionated and immunoblotted with anti-STAT3 and anti-SH2B1 antibodies. α-Tubulin was used as a marker for the cytoplasmic fraction and lamin B as a marker for the nuclear fraction. Cytoplasmic STAT3 was normalized to cytoplasmic α-tubulin, and nuclear STAT3 was normalized to nuclear lamin B levels. Values are means ± SEM from three independent experiments. (E) COS7 cells were transiently transfected with GFP-SH2B1β, GFP-SH2B1β(ΔNES), or GFP-SH2B1β(ΔNLS) along with pm67⁴ and a Renilla luciferase plasmid. The firefly luciferase activities were normalized to the corresponding Renilla luciferase activities. Values are the means ± SEM from three independent experiments. *, P < 0.05 by paired Student's t test.