Shoc2 depletion reduces phospho-ERK and phospho-MEK levels induced by RTK activation.

<p>HEK293T cells were transiently transfected (100 nM) with either control siRNA (siRNA-Control) or human Shoc2-specific siRNA (siRNA-Shoc2). At 48 h post-transfection, cells were serum-starved for 18 h and then incubated with vehicle (0), or 5-25 ng/ml FGF for 10 min. (<b>A</b>), endogenous Shoc2 expression was assessed, from the cell lysates, by immunoblot using anti-Shoc2 rabbit polyclonal antibody and normalized to tubulin levels, expressed as a percentage (corresponding to the mean of five separate assays, in all cases with a SD ≤10% of the mean). The specific effect of the siRNA-Shoc2 was tested throughout detection of Grb2, Ras, C-Raf, ERK and MEK protein levels, by immunoblot of the cell lysates with the corresponding antibodies. Cell lysates were also analyzed by immunoblot using anti-p-ERK/ERK, and anti-p-MEK/MEK antibodies (bottom panels); in each case, fold increase denotes the ratio p-ERK/ERK, and p-MEK/MEK levels estimated as the mean of five separate assays (in all cases with a SD ≤10% of the mean). (<b>B</b>), the expression of Shoc2 was assessed by qRT-PCR. Data represents the mean ± SD of four independent experiments. The detection of GAPDH (normalization control) was performed using specific primers. Results were calculated by the 2-^ΔΔCT method. *P≤0.001 compared with siRNA-control; bars show SD.</p

Shoc2 overexpression increases EGF-induced PC12 differentiation.

<p><b>A</b>), PC12 cells were co-transfected with plasmids pCEFL-KZ-GFP (0.3 µg) and pCEFL-KZ-AU5 (0.7 µg) (top: GFP + EGF) or pCEFL-KZ-GFP (0.3 µg) and pCEFL-KZ-AU5-hShoc2 (0.7 µg) (bottom: Shoc2-GFP + EGF). Transfected PC12 cells were stimulated with EGF (100 ng/ml) for 72 h, fixed, and the presence of extending neurites was analyzed in GFP-positive cells by fluorescence microscopy (right: white arrows). Total cells were visualized with transmitted-light (left). A process equal in length or greater than one cell body was considered a neurite; bars, 10 µm. PC12 cells co-transfected with pCEFL-KZ-GFP and pCEFL-KZ-AU5-hShoc2 but not stimulated with EGF or NGF did not have neurites (not shown). <b>B</b>), PC12 transfected cells (as in A) were stimulated with EGF (100 ng/ml) for 72 h, fixed. Cells were incubated with anti-AU5 monoclonal antibody for 1h at room temperature and, after several washes, were incubated with Alexa 488-conjugated secondary antibodies. The presence of extending neurites was analyzed with transmitted-light (left-picture). PC12 cells overexpressing exogenous Shoc2 were detected as AU5-positive cells by fluorescence microscopy (right-picture and white arrows). A process equal to or greater than one cell body in length was considered a neurite; bars, 10 µm. <b>C</b>), histograms represent the percentage of transfected PC12 cells (GFP-positive: GFP vs Shoc2-GFP) with neurites after EGF treatment of panel A; values are the mean of three separate assays, in which at least 50 GFP-positive cells/assay were analyzed (*P<0.01); bars show SD.</p

Shoc2 overexpression enhances the intensity and/or duration of RTK-elicited ERK activation.

<p>HEK293T cells transiently co-transfected with 1 µg pCEFL-KZ-HA-ERK1 and 2 µg pCEFL-KZ-AU5-hShoc2 (S) or 2 µg pCEFL-KZ-AU5 (-) were serum-starved for 18 h and then incubated with vehicle (0), 25 ng/ml FGF (<b>A</b>), or 10 ng/ml EGF (<b>B</b>), for 5 to 240 min. Cell lysates were immunoprecipitated with anti-HA antibody and analyzed by immunoblot using anti-p-ERK and -HA antibodies, to determine p-ERK1 and total HA-ERK1. Fold increase denotes the ratio p-ERK/ERK levels estimated as the mean of three separate assays (in all cases with a SD ≤10% of the mean). Expression levels of transfected AU5-hShoc2 constructs were detected by immunoblotting whole cell extracts (WCL) with the appropriate anti-AU5 antibody, and total Shoc2 expression was assessed by re-blotting of the same filters against anti-Shoc2 rabbit polyclonal antibody and using tubulin detection as control. In each point-time, the protein levels of Shoc2 were normalized versus tubulin amount and vector sample (-). Results were similar in three independent experiments.</p

Shoc2 knockdown reduces NGF-elicited of PC12 cell differentiation.

<p>A), PC12 cells were nucleofected with shRNA-control-GFP plasmid (ShRNA Control) or specific rat shRNA-Shoc2-GFP plasmids (ShRNA Shoc2 #1-3). At 48 h post-transfection, cell lysates were analyzed by immunoblot. B), Shoc2 levels determined using anti-Shoc2 polyclonal antibody were normalized to total ERK levels, and expressed as a percentage (right). Results were similar in two additional experiments. *P≤0.001 and n.s. (not significant) compared with siRNA-control; bars show SD. C), PC12 cells nucleofected with either shRNA-control-GFP plasmid (ShRNA Control) or specific rat shRNA-Shoc2-GFP plasmids (ShRNA Shoc2 (2) and (3)) were stimulated with 100 ng/ml NGF for 72 h. After fixing, GFP-positive cells were detected by fluorescence microscopy (right: arrows) and total cells were visualized with transmitted light (left). Extending processes equal in length or greater than two cell bodies were considered neurites; bars, 10 µm. D), histograms represent the percentage of transfected PC12 cells (GFP-positive) with neurites after NGF treatment; values are the mean of three separate assays, in which at least 50 GFP-positive cells/assay were analyzed (*ShRNA Shoc2 (2 or 3) vs ShRNA Control; P<0.01), bars show SD.</p

Shoc2 knockdown reduces NGF-induced ERK activation in PC12 cells.

<p>PC12 cells nucleofected with shRNA-control-GFP plasmid (ShRNA Control) or specific rat shRNA-Shoc2-GFP #3 plasmid (ShRNA Shoc2) were stimulated with 100 ng/ml NGF for the times indicated. In each assay, 2 µg PC12 cell extracts were analyzed by xMAP technology in a Luminex-200 platform for quantitative and simultaneous detection of phospho-ERK (pTpY183/185) and total ERK protein (Panel <b>A</b>), phospho-AKT and total AKT (Panel <b>B</b>) or phospho-GSK3 and total GSK3 protein (Panel <b>C</b>). Histograms show the ratio of p-ERK/ERK (Panel <b>A</b>), p-AKT/AKT (Panel <b>B</b>) or p-GSK3/GSK3 levels (Panel <b>C</b>) after the indicated time of NGF stimulation; values are the mean of three separate assays performed in duplicate (*P≤0.001), bars show SD.</p