Glucose Regulation of Thrombospondin and Its Role in the Modulation of Smooth Muscle Cell Proliferation
Figure 1
TSP-1 levels correlate with the level of intact IAP in SMC. (a) SMC were grown to confluency in DMEM containing 5 or 25 mM glucose and then incubated overnight in SFM with the appropriate level of glucose. Extracellular matrix, membrane extract or conditioned medium was harvested as described in the methods. Equal amounts of protein were immunoblotted (IB) with either an anti-TSP-1 or β3 antibody. (b) SMC were grown and incubated overnight in SFM as described for (a). The following day the incubation was continued for a further 6 hours after the addition (+) of TSP-1, (1 g/mL). The graphs show arbitrary scanning units derived from the immunoblots with the anti-IAP antibody from three independent experiments. when the level of IAP in the presence of TSP-1 is compared to SFM alone. (c) Equal amount of protein from the homogenates of aorta from normal (Con) and hyperglycemic (STZ) pigs were separated by SDS PAGE and the level of TSP-1, IAP and the IGF-IR determined by western immunoblotting (IB). The graphs shows show arbitrary scanning units derived from the immunoblots with the anti-TSP-1 or IAP antibody, as labeled, from three independent experiments. when the level of TSP-1/IAP from the STZ animals is compared with control. (d) SMC were grown and incubated overnight in SFM as described for (a). The following day the incubation was continued for a further 6 hours after the addition (+) of 4N1K or 4N1G, (1 g/mL). The graph shows the fold difference in IAP levels compared with the level derived from SMC incubated in 25 mM glucose using arbitrary scanning units from the immunoblots with the anti-IAP antibody from three independent experiments.
