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Fig. 4. Laminin, IGF-1 and BDNF stimulate transient phosphorylation of SIRP
and its association with SHP-2 in GCPs. (A) GCPs with or without stimulation were immunoprecipitated with anti-SIRP antibody after 1 minute of incubation. Immunoprecipitates were analyzed by western blot with anti-pTyr and anti-SHP-2 to examine SIRP phosphorylation and SHP-2 co-immunoprecipitation, respectively (=anti). The bar graphs show quantitation of SIRP phosphorylation (left) and of SHP-2 co-immunoprecipitation (right), expressed as the percentage change over control level. Results are means ± s.e.m. of at least four independent experiments. *P
0.01 compared to control. (B) The same experiment as in A, but 5 minutes incubation time. The bar graphs show decreases in phosphorylation and SHP-2 co-immunoprecipitation relative to control. (C,D) Effects of Src family kinase inhibitor on laminin-, IGF-1- and BDNF-stimulated tyrosyl phosphorylation (C) and SHP-2 binding of SIRP (D). The Src kinase inhibitor, PP2, was added to GCPs prior to incubation in control or stimulated conditions. PP3 added in control conditions served as a negative control for PP2. After 1 minute incubation, GCPs were solubilized and SIRP immunoprecipitated. Western blots of SIRP immunoprecipitates were probed with anti-pTyr to examine SIRP phosphorylation (C) or with anti-SHP-2 to reveal SHP-2 co-immunoprecipitation (D). In addition, they were probed with anti-SIRP to control for SIRP loading. Note that PP2, even with stimulation, reduced SIRP phosphorylation and co-immunoprecipitation of SHP-2 to below control levels without PP2 treatment (C and D) or with PP3 treatment (C). The numbers below the blots are mean ratios ± s.e.m. (n=2 in C; n=3 in D) of pTyr or SHP-2 label over SIRP label (arbitrary units, normalized to control without PP2 treatment).
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