Inhibition of Aberrant IGF-I Signaling in Diabetic Male Rat Retina Prevents and Reverses Changes of Diabetic Retinopathy

Figure 2

Disruption of IAP/SHPS-1 association attenuated IGF-I downstream signaling and IGF-I-stimulated cell proliferation. Rat endothelial cells were grown to confluency in high-glucose (HG, 25 mM) medium then placed in serum-free HG medium. The indicated concentration of anti-IAP antibody was incubated with the cells for 2 h, and IGF-I (100 ng/ml) was added for 10 min before the cells were harvested. (a) Cell lysates were immunoprecipitated with an anti-pY99 antibody and immunoblotted with an anti-Shc antibody. The same amount of cell lysate was immunoblotted with an anti-Shc antibody. The bar graph shows the of scanning densitometry values of phospho-Shc divided by total Shc. indicates the significant differences. The cell lysates were also immunoblotted with anti-pErk1/2 or Erk1/2 antibody (b) and anti-pAKT or AKT antibody (c). The bar graph shows the of scanning densitometry values of pErk1/2 divided by total Erk1/2 (b) or pAkt divided by total Akt (c). and indicate the significant differences. (d) Cell proliferation assays were performed following a procedure described in Materials and Methods. indicates the significant difference between two treatments. The results are the of three separate experiments with 3 determinations of each data point in each experiment. (e) Cell lysate was immunoblotted with anti-VEGF or β-actin antibody. The bar graph shows the of scanning densitometry values of VEGF divided by β-actin. indicates a significant difference. Each experiment was repeated 3 times, and the results were similar to the representative immunoblots shown in the figures.

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