Identification of Compounds That Inhibit IGF-I Signaling in Hyperglycemia
Figure 2
Acceleration of IAP cleavage inhibits IAP-SHPS-1 association and SHPS-1 phosphorylation. SMCs were grown to confluency in DMEM containing 25 mM glucose and then incubated overnight in SFM. They were then incubated in HG-SFM or HG-SFM containing the test compounds (mM) for 6 hours. Where indicated the cultures were treated with IGF-I (100 ng/mL) for 5 minutes prior to lysis. Cell lysates were then immunoprecipitated and proteins visualized by western immunoblotting. To control for differences in total protein samples were also immunoblotted with an anti-SHPS-1 antibody. (a) IAP association with SHPS-1 was determined following immunoprecipitation (IP) with an anti-SHPS-1 antibody and immunoblotting (IB) with the anti-IAP monoclonal antibody, B6H12. (b) SHPS-1 phosphorylation in response to IGF-I was determined by IP with an anti-SHPS-1 antibody and IB with an antiphosphotyrosine antibody (p-Tyr). To control for differences in total protein samples were also immunoblotted with an anti-SHPS-1 antibody.
