Figure 8.
Representative micrographs and results of densitometric evaluations of multiple Western blots with anti-phospho-Akt (A) and anti-phospho-Erk1/Erk2 (B) antibodies demonstrate that pretreatment of AoSMCs with Nase, which caused desialylation of the β subunits of their PDGF and IGF-1 receptors, also resulted in the inhibition of downstream Akt and Erk1/Erk2 phosphorylation normally induced by their respective ligands, IGF-2 and PDGF-BB. Quadruplicate cultures of AoSMCs derived from three different individuals were preincubated for 30 minutes in the presence or absence of 50 mU/ml of exogenous Nase and then exposed for 10 minutes to IGF-2 (100 ng/ml) or PDGF (10 ng/ml). Cells were then lysed in 2× sample buffer. The lysate aliquots were subjected to SDS-PAGE, transferred to nitrocellulose, and subsequently immunoblotted with the indicated antibodies. In each experiment the parallel Western blots were first probed with antibodies against phospho-Akt or phospho-Erk1/2 and, respectively reprobed with anti-Akt or anti-Erk1/2 antibodies. Loadings of equivalent amounts of protein were additionally confirmed by the immunodetection of β-actin. The quantitative densitometric evaluations of phospho-Akt and Phospho-Erk1/Erk2 immunoblots were normalized against their immunodetectable non-phosphorylated forms. The final results are presented in arbitrary units and reflect the proportional change from the untreated control.