SH2B1β interacts with TrkB.

<p>Lysates from 293T cells co-transfected with myc-SH2B1β and TrkB followed by 50 ng/ml BDNF stimulation for 0, 10, 30, 60 minutes were collected and subjected to immunoprecipitation using anti-myc antibody followed by immunoblotting with anti-myc, TrkB or pTrkB(Y706) antibody. IgG was used as negative control. Representative blots are shown from two independent experiments.</p

Knock-down of SH2B1 expression reduces BDNF-induced neurite outgrowth in PC12-TrkB cells.

<p>(A) PC12-TrkB stable cell line and parental PC12 cells were treated with 50 ng/ml BDNF in low serum medium for 0, 1 or 3 days. Representative live cell images are shown. Scale bar  =  20 µm. (B) PC12 cells expressing TrkB and shLacZ (PC12-TrkB+shLacZ) or TrkB and shSH2B1 (PC12-TrkB+shSH2B1) were established. Cell lysates from PC12-TrkB+shLacZ and PC12-TrkB+shSH2B1 cells were collected and analyzed via SDS-PAGE and immunoblotted with anti-SH2B1 or α-tubulin antibody. α-tubulin was used as a loading control. (C) PC12-TrkB+shLacZ cells (i, iii, v) and PC12-TrkB+shSH2B1 cells (ii, iv, vi) were treated with 100 ng/ml BDNF in low serum medium for 0 (i, ii), 1 (iii, iv) or 3 (v, vi) days. Representative live cell images are shown. Scale bar  =  50 µm. (D) The percentages of neuronal differentiation for PC12-TrkB+shLacZ and PC12-TrkB+shSH2B1 cells treated with BDNF for 1 or 3 days were quantified.</p

Overexpressing SH2B1β enhances BDNF-induced neurite outgrowth and branching in hippocampal neurons.

<p>(A) E18 primary hippocampal neurons were transiently transfected with either GFP or GFP-SH2B1β on DIV 4. One day after transfection, neurons were treated with 50 ng/ml BDNF for 2 days. The morphology of the neurons was visualized on DIV 7 by Zeiss LSM510 meta confocal microscope using 20X (NA/0.75) objective. Boxes mark the neurites of hippocampal neurons. Enlarged images of the neurites and branching are shown on the right panels. Scale bar  =  10 µm. (B) Neurite length of the transfected neurons were measured as described in the Materials and Methods. Average length of the longest neurite per neuron. The length of neurites were measured using Simple Neurite Tracer plug-in of the ImageJ software. (*: P<0.05, compared with GFP without BDNF). (C) The number of end-points were quantified by ImageJ software. 15–20 GFP- or GFP-SH2B1β-transfected hippocampal neurons were counted from three independent experiments. Values are mean ± SE from three independent experiments and statistically compared by student’s <i>t</i>-test (*: P<0.05).</p

BDNF-induced phosphorylation of ERK1/2 and AKT are enhanced by overexpression of SH2B1β.

<p>(A) PC12-GFP+TrkB or PC12-SH2B1β+TrkB cells were cultured in serum-free medium overnight before stimulation of 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 anti-pERK1/2, ERK1/2, pAKT(S473), AKT, pPLCγ1(Y783), and PLCγ1 antibodies. Representative blots from three independent experiments are shown. (B) pERK1/2, pAKT(S473), and pPLCγ1(Y783) levels were normalized to total ERK1/2, AKT and PLCγ1, respectively. The relative pERK1/2, pAKT(S473) and pPLCγ1(Y783) levels for the 5 min time point of PC12-SH2B1β+TrkB cells were used as 1. The error bars represent S.E.M. from three independent experiments. (*: p<0.05, paired Student’s t-test)</p

SH2 domain of SH2B1β is required to enhance BDNF-induced neurite outgrowth and signaling.

<p>PC12 cells overexpressing GFP and TrkB (PC12-GFP+TrkB), GFP-SH2B1β and TrkB (PC12-SH2B1β+TrkB) or GFP-SH2B1β(R555E) and TrkB (PC12-R555E+TrkB) were established. (A) Cell lysates from PC12-GFP+TrkB, GFP-SH2B1β and PC12-R555E+TrkB cells were analyzed via SDS-PAGE and immunoblotted with anti-SH2B1 or TrkB antibody to determine the expression of GFP-SH2B1β, GFP-SH2B1β(R555E), endogenous SH2B1 and TrkB. The relative expression levels of GFP-SH2B1β and GFP-SH2B1β(R555E) were normalized to the respective endogenous SH2B1 level in each cell line. (B) PC12-GFP+TrkB cells (i, iii, v), PC12-SH2B1β+TrkB cells (ii, iv, vi) or PC12-R555E+TrkB cells (vii, viii, ix) were treated with 100 ng/ml BDNF in low serum medium for 0 (i, iv, vii), 1 (ii, v, viii) or 3 (iii, vi, ix) days. Representative images of live cells are shown. Scale car  =  50 µm. (C) The percentage of differentiated cells for PC12-GFP+TrkB, PC12-SH2B1β+TrkB or PC12-R555E+TrkB cells treated with BDNF for 1 or 3 days was counted. Values are means ± S.E.M. from three independent experiments (*, <b>#</b>: p<0.05, paired Student’s t-test. *: comparison between PC12-GFP+TrkB and PC12-R555E+TrKB cells for the same BDNF treatment day; <b>#</b>: comparison between PC12-SH2B1β+TrkB and PC12-R555E+TrKB cells for the same BDNF treatment day). (D) PC12-GFP+TrkB or PC12-R555E+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 anti-pERK1/2, ERK1/2, pAKT(S473), AKT, pSTAT3(S727) and STAT3 antibodies. Representative blots from three independent experiments are shown. pERK1/2, pAKT(S473), and pSTAT3(S727) level were normalized to total ERK1/2, AKT, and STAT3, respectively and the relative pERK1/2, pAKT(S473), and pSTAT3(S727) levels for the 5 min time point of PC12-GFP+TrkB cells were used as 1. The error bars represent S.E.M. from three independent experiments. (*: p<0.05, paired Student’s t-test).</p

Overexpression of SH2B1β enhances BDNF-induced neurite outgrowth.

<p>PC12 cells overexpressing GFP and TrkB (PC12-GFP+TrkB) or GFP-SH2B1β and TrkB (PC12-SH2B1β+TrkB) were established. (A) Cell lysates from PC12-GFP+TrkB and PC12-SH2B1β+TrkB cells were collected and analyzed via SDS-PAGE and immunoblotted with anti-SH2B1, TrkB and α-tubulin antibodies. α-tubulin was used as a loading control. (B) PC12-GFP+TrkB cells (i, iii, v) and PC12-SH2B1β+TrkB cells (ii, iv, vi) were treated with 100 ng/ml BDNF in low serum medium for 0 (i, ii), 1 (iii, iv) or 3 (v, vi) days. Representative live cell images are shown. Scale bar  =  50 µm. (C) The percentages of neuronal differentiation for PC12-GFP+TrkB and PC12-SH2B1β+TrkB cells treated with BDNF for 1 or 3 days were quantified. Values are means ± S.E.M. from three independent experiments. (*: compared to the percentages of differentiated PC12-GFP+TrKB cells for the same BDNF treatment day, p<0.05, paired Student’s t-test). (D) E18 cortical neurons were transiently transfected with myc-SH2B1β on DIV 10. One day after transfection, neurons were treated with 50 ng/ml BDNF for 0, 10, 30, 60 mins. Cell lysates were collected and subjected to immunoprecipitation using anti-myc antibody followed by immunoblotting with anti-SH2B1 or anti-pTyr (4G10) antibody.</p

The tyrosine phosphorylation of SH2B1β contributes to SH2B1β-mediated enhancement of BDNF-induced neurite outgrowth and signaling.

<p>(A) Cell lysates from PC12-GFP+TrkB, PC12-SH2B1β+TrkB and PC12-9YF+TrkB cells were analyzed via SDS-PAGE and immunoblotted with anti-SH2B1 or TrkB antibody to determine the expression of GFP-SH2B1β, GFP-SH2B1β(9YF), endogenous SH2B1 and TrkB. (B) PC12-GFP+TrkB cells (i, ii, iii), PC12-SH2B1β+TrkB cells (iv, v, vi) or PC12-9YF+TrkB cells (vii, viii, ix) were treated with 100 ng/ml BDNF in low serum medium for 0 (i, iv, vii), 1 (ii, v, viii) or 3 (iii, vi, ix) days. Representative live cell images are shown. (C) The percentage of differentiated cells in PC12-GFP+TrkB, PC12-SH2B1β+TrkB cells or PC12-9YF+TrkB was counted after BDNF treatment for 1 or 3 days. Values were means ± S.E.M from three independent experiments. (*: compared to the percentage of PC12-9YF+TrkB cells for the same day of BDNF treatment, p<0.05, paired Student’s t-test; #: compared to the percentage of PC12-GFP+TrkB cells for the same BDNF treatment day, p<0.05, paired Student’s t-test) (D) PC12-GFP+TrkB or PC12-9YF+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 anti-pERK1/2, ERK1/2, pAKT(S473), AKT, pPLCγ1(Y783), PLCγ1, pSTAT3(S727), and STAT3 antibodies. Representative blots from two independent experiments are shown. (E) PC12-SH2B1β+TrkB or PC12-9YF+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 anti-pERK1/2, ERK1/2, pAKT(S473), AKT, pPLCγ1(Y783), PLCγ1, pSTAT3(S727) and STAT3 antibodies. Representative blots from two independent experiments are shown.</p

Reduction of SH2B1 expression attenuated BDNF-induced neurite outgrowth of cortical neurons.

<p>Cultured cortical neurons on DIV 4 were transfected with the shLacZ or shSH2B1 together with pEGFP vector. Twenty-four hours after transfection, cells were either untreated or treated with 50 ng/ml BDNF for 1 day. (A) Representative live cell images of primary cortical neurons are shown using Zeiss Observer Z1 microscope using 10X (NA/0.3). Scale bar  =  50 µm. (B) Average length of the longest neurite per neuron. 20–35 GFP-transfected cortical neurons were counted per condition for each experiment. Values were means ± S.E.M from three independent experiments (*: p<0.05, paired Student’s t-test). (C) Average number of end-points per neuron. 10–15 GFP-transfected cortical neurons were counted per condition for each experiment. Values were means ± S.E.M from three independent experiments (*: p<0.05, paired Student’s t-test). The length and branches of neurites were measured using ImageJ software as described in the Materials and Methods section.</p

MEK-ERK1/2 and PI3K-AKT signaling pathways are required for SH2B1β-enhanced neurite outgrowth.

<p>(A) PC12-GFP+TrkB or PC12-SH2B1β+TrkB cells were incubated in serum-free medium overnight and 20 µM U0126 was added to cells 1 h before 5 min stimulation of 50 ng/ml BDNF. Equal amount of proteins from the cell lysates was resolved with SDS-PAGE and immunoblotted with anti-pERK1/2 antibody. (B) PC12-GFP+TrkB cells (i, iii, v, vii) or PC12-SH2B1β+TrkB cells (ii, iv, vi, viii) were pre-treated with 20 µM U0126 (iii, iv, vii, viii) for 1 h, followed by 50 ng/ml BDNF stimulation in low serum medium. The neurite outgrowth was monitored for 3 days. Representative images of live cells are shown. (C) The percentages of BDNF-induced neuronal differentiation of mock- or U0126-treated PC12-GFP+TrkB and PC12-SH2B1β+TrkB cells were quantified. Values are means ± S.E.M. from three independent experiments. (*: p<0.05, paired Student’s t-test) (D) PC12-GFP+TrkB or PC12-SH2B1β+TrkB cells were incubated in serum-free medium overnight and 20 µM LY294002 was added to cells 1 h before 5 min stimulation of 50 ng/ml BDNF. Equal amount of cell lysates was resolved with SDS-PAGE and immunoblotted with anti-pAKT(S473) antibody. (E) PC12-GFP+TrkB cells (i, iii, v, vii) or PC12-SH2B1β+TrkB cells (ii, iv, vi, viii) were pre-treated with 20 µM LY294002 (iii, iv, vii, viii) for 1 h, followed by 50 ng/ml BDNF stimulation in low serum medium. The neurite outgrowth was monitored for 3 days. Representative images of live cells are shown. (F) The percentages of BDNF-induced neuronal differentiation of mock- or LY294002-treated PC12-GFP+TrkB and PC12-SH2B1β+TrkB cells were quantified. Values are means ± S.E.M. from three independent experiments. (*: p<0.05, paired Student’s t-test)</p

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

<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