SH2B1β enhances BDNF-induced pSTAT3(S727) through MEK-ERK1/2 and PI3K-AKT signaling pathways.

Abstract

<p>(A) PC12-GFP+TrkB or PC12-SH2B1β+TrkB cells were incubated in serum-free medium overnight before stimulation with 50 ng/ml BDNF for the indicated time points. Lysates were collected and equal amount of proteins was separated by SDS-PAGE and immunoblotted with either anti-pSTAT3(S727) or anti-STAT3 antibody. Representative blots from three independent experiments are shown. pSTAT3(S727) level was normalized to total STAT3 in PC12-GFP+TrkB and PC12-SH2B1β+TrkB cells both treated with BDNF and the relative pSTAT3(S727) level for the 5 min time point of PC12-SH2B1β+TrkB cells was used as 1. The error bars represent S.E.M. from three independent experiments. (*: p<0.05, paired Student’s t-test) (B) Cells were incubated in serum-free medium for 16 h, then U0126 (20 µM) was added to cells 1 h before 50 ng/ml BDNF stimulation for 5 min. Cell lysate was collected and equal amount of proteins was analyzed with SDS-PAGE and immunoblotted with anti-pSTAT3(S727) antibody. pSTAT3(S727) level was normalized to total STAT3 in PC12-GFP+TrkB and PC12-SH2B1β+TrkB cells both treated with either BDNF or U0126 and the relative pSTAT3(S727) level for the 5 min time point of BDNF stimulation in PC12-SH2B1β+TrkB cells was used as 1. The error bars represent S.E.M. from free independent experiments. (*: P<0.05, paired Student’s t-test) (C) PC12-GFP+TrkB or PC12-SH2B1β+TrkB cells were treated with 20 µM LY294002 for the indicated time points and processed as in (B). Lysates were collected and equal amount of proteins was separated by SDS-PAGE and immunoblotted with either anti-pSTAT3(S727) or anti-STAT3 antibody. Representative blots from three independent experiments are shown. The relative pSTAT3(S727) level was normalized as described in (B). The error bars represent S.E.M. from three independent experiments. (*: P<0.05, paired Student’s t-test).</p