Fig. 3.

g-OxLDL enhances IGF-I-stimulated SHPS-1 phosphorylation and Shc recruitment to SHPS-1. SMC were grown to confluency in medium containing 5 mm glucose before overnight incubation in SFM. g-OxLDL (5 μg/ml) or n-LDL (5 μg/ml) was added for 4 h. IGF-I was added (100 ng/ml) for 5 min at the end of the 4-h incubation before lysing of the cells. A, SHPS-1 phosphorylation was determined by immunoprecipitating (IP) with an anti-SHPS-1 antibody and immunoblotting with an anti-phosphotyrosine antibody (p-Tyr). Equal amounts of cell lysates were also immunoprecipitated with the anti-SHPS-1 antibody and immunoblotted with the same antibody to demonstrate that the difference in response was not due to different amounts of SHPS-1 protein. The graph shows the increase in SHPS-1 phosphorylation in response to IGF-I (mean ± sem, n = 3). **, P < 0.01 when SHPS-1 phosphorylation in response to IGF-I in the presence of g-OxLDL is compared with the response to IGF-I alone. B, The extent of Shc association with SHPS-1 was determined by immunoprecipitating (IP) cell lysates with an anti-Shc antibody and then immunoblotting with an anti-SHPS-1 antibody. Equal amounts of cell lysates were also immunoprecipitated with the anti-Shc antibody and immunoblotted with the same antibody. The graph shows the increase in Shc association with SHPS-1 in response to IGF-I (mean ± sem, n = 3). **, P < 0.01 when Shc recruitment to SHPS-1 in response to IGF-I in the presence of g-OxLDL is compared with the response to IGF-I alone. The results shown are representative of three similar experiments performed independently.