Journal: Oncogene
Article Title: Domain-specific function of ShcC docking protein in neuroblastoma cells.
doi: 10.1038/sj.onc.1208523
Figure Lengend Snippet: Figure 6 Anchorage-independent activation of Src family kinases (SFKs) and tyrosine phosphorylation of Cas in NB-39-nu is affected by the expression of ShcC. The cell suspension culture of ShcC mutant cells was essentially performed as described in Materials and methods. A: attached cells. S: cells in a suspended condition. Lysates were duplicated and detected by immunoprecipitation and Western analysis. (a, b and c) Cells cultured for 24 h in adherent or suspended conditions were analysed with the antibodies against each Src family kinase shown in the figure. Anti-phospho-Src Y416 recognizes all Src family members phosphorylated at the tyrosine corresponding to Tyr416 of avian Src. (d) Same analysis performed as (a) (b) and (c) using the antibodies against for phospho-460Y of Cas (aP-Cas460Y) and Cas protein (aCas). (e) Time course of tyrosine phosphorylation of Cas in each ShcC mutant cells cultured in suspension for the indicated time periods. (f) Effect of Src inhibitor, PP2, on the tyrosine phosphorylation of Cas in NB-39-nu control cells. The cells cultured for 24 h were harvested following treatment with 10 mM of PP2 for 2 h. As a negative control, the same dose of PP3 was used in place of PP2. (g) ShcC forms complex with SFKs following the stimulation of fibronectin. Lysates of mock cells and ShcC-wt cells were analysed after the stimulation with EGF or fibronectin as described in Materials and methods. -: serum free without stimulation; E: stimulated by EGF; F: stimulated by fibronectin. Ig: immunoglobulin. Anti-pan-Src antibody (SRC2) reacts with c-Src p60, Yes p62, Fyn p59, c-Fgr p55 and c-Src2. closed arrowhead: SFK; open arrowhead: p52 ShcC
Article Snippet: Other antibodies were purchased as follows: anti-phosphotyrosine antibody (4G10) (Upstate Biotechnology, Inc), anti-p44/42 MAPK (ERK1/2) and anti-phospho-p44/42 MAPK (phospho-ERK1/2) antibodies (BioLabs), anti-Akt and anti-phospho-Akt (Ser473) antibodies (Cell Signaling), anti-c-Src antibody (Upstate Biotechnology, Inc.), anti-phospho Src family (Tyr416) antibody (Cell Signaling), anti-Fyn antibody and anti-pan-Src antibody (SRC2) (SantaCruz Biotechnology, Inc.).
Techniques: Activation Assay, Phospho-proteomics, Expressing, Suspension, Mutagenesis, Immunoprecipitation, Western Blot, Cell Culture, Control, Negative Control
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