Neuropilin 1 Mediates Keratinocyte Growth Factor Signaling in Adipose-Derived Stem Cells: Potential Involvement in Adipogenesis

Figure 9

Effect of NRP1 silencing on KGF-mediated phosphorylation of ERK, p38, and Akt. (a) ASCs transfected with NRP1-specific siRNA (siNRP) or nonspecific control siRNA (siNC), treated or not with 20 ng/ml KGF for 30 min at 37°C, were lysed, and NRP1 expression was analyzed by immunoblotting with anti-NRP1 antibodies. siNRP induced a marked reduction in NRP1 expression in both untreated and KGF-treated cells. Western blot with anti-tubulin antibodies was used as loading control. (b) The same lysates were analyzed by immunoblotting with anti-phospho-ERK antibody. Transfection with siNRP significantly inhibits ERK phosphorylation induced by KGF treatment. The levels of total ERK were assessed by Western blot with anti-ERK1/2 antibodies. (c) Phosphorylation of p38 was assessed by Western blot analysis with a phospho-specific p38 monoclonal antibody (pp38). Levels of total p38 were assessed by blotting with a p38-specific antibody and served as loading control. (d) Phosphorylation of Akt was assessed by Western blot analysis with a phospho-specific Akt monoclonal antibody (pAkt). Levels of total Akt were assessed by blotting with an Akt-specific antibody and served as loading control.The intensity of the bands was evaluated by densitometric analysis; the values from a representative experiment were normalized, expressed as fold increase with respect to the untreated siNC sample and reported as a graph. and .

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