Identification of Compounds That Inhibit IGF-I Signaling in Hyperglycemia

Figure 1

Acceleration of IAP cleavage by positive hit compounds (a) and (b). SMCs were grown to confluency in DMEM containing 25 mM glucose and then incubated overnight in SFM. They were then incubated in HG-SFM alone or HG-SFM containing the test compounds (2, 5 or 10 𝜇M) for 6 hours. Following lysis and separation by SDS-PAGE the amount of intact IAP was detected by western immunoblotting with the anti-IAP monoclonal antibody, B6H12 which recognizes intact and residual membrane associated IAP fragment or the anti-IAP antibody R569 which specifically recognizes intact IAP. To control for differences in protein equal amounts of lysate were also immunoblotted with the anti-SHPS-1 antibody. (c) REC were maintained in medium containing 25 mM glucose prior to incubation overnight in SFM. The test compounds were added at a concentration of 5 𝜇M for 6 hours. IAP was visualized as described above.