Glucose Regulation of Thrombospondin and Its Role in the Modulation of Smooth Muscle Cell Proliferation
Figure 4
Restoration of the IGF-I response by addition of the CD47/IAP binding domain of TSP-1. (a) SMCs expressing either the TSP-1 or a scrambled (Scr) RNAi construct were grown to confluency in DMEM containing 25 mM glucose and then incubated overnight in SFM with 25 mM glucose. After overnight incubation in SFM cells were incubated for 6 hours with 4N1K (1 g/mL) prior to lysis. The amount of intact IAP in each lysate was determined by immunoblotting with the anti-IAP antibody specific for intact IAP and the association between IAP and SHPS-1 was determined by coimmunoprecipitation. The graphs shows the results derived from western immunoblots from three similar experiments expressed as arbitrary scanning units increase ( when the amount in the TSP-1 lysate is compared with the Scr cell lysate). (b) SMCs expressing the TSP-1 RNAi construct were grown to confluency in DMEM containing 25 mM glucose and then incubated overnight in SFM with 25 mM glucose. After overnight incubation in SFM cells were incubated for 6 hours with 4N1K (1 g/mL) then treated with IGF-I (+) where indicted for 5 minutes prior to lysis. SHPS-1 and Shc phosphorylation in equal amounts of cell lysate was determined by immunoprecipitation and immunoblotting with an antiphosphotyrosine antibody (PY99). The graphs shows the results derived from western immunoblots from three similar experiments expressed as fold increase in phosphorylation after treatment with IGF-I ( when the phosphorylation in the presence of 4N1K is compared with the absence). (c) cells grown 25 mM were plated in each well of a 24 well plate prior to exposure to 4N1K (1 g/mL) and IGF-I (100 ng/mL) in DMEM 0.2% platelet poor plasma. 48 hours after the addition of IGF-I cell number was determined by trypan blue staining and counting. when cell number in response to IGF-I is compared with control.
