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

Figure 8

Effect of NRP1 silencing on KGF-mediated phosphorylation of ERK. Expression of NRP1 assessed by PCR (a) and Western blot analysis (b) on ASC whole cell lysates. MDA-MB-231 cells were used as positive control for NRP1 expression. GAPDH mRNA expression and blotting with anti-tubulin antibody served as loading control for PCR and Western blot analysis, respectively. The images are representative of at least three independent experiments. (c) Coimmunoprecipitation assay was performed to study in vivo interaction between KGF and NRP1 proteins. ASCs, untreated or treated with 20 ng/ml KGF for 5 min, were immunoprecipitated with anti-NRP1 antibody and blotted with anti-FGF7 antibody. Western blot with anti-NRP1 antibody was used as loading control. (d) ASCs transfected with NRP1-specific siRNA (siNRP) or nonspecific control siRNA (siNC), treated or not with 20 ng/ml KGF for 5 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. (e) 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. 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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