Figure 3. c-Kit inhibits NF-κB activity in smooth muscle cells (SMC) through the actions of TAK1 and NLK.

(A) Protein expression of the TAK1 and NLK regulatory proteins in c-Kit deficient (Kit^W∕W−v), control (Kit^+∕+), and c-Kit rescued SMC (Kit^R) as determined by Western blot. Protein expression is expressed as the mean ± standard deviation (SD) of the TAK1∕β-actin and NLK∕β-actin signal ratios normalized with respect to the control group (Kit^+∕+) (n = 3 per cell type). (B–C) Protein expression of TAK1 (B) and NLK (C) in Kit^+∕+ cells transduced with lentivirus-encoded siRNAs of the corresponding targets or GFP control. Protein expression is expressed as the mean ± SD of the TAK1∕β-actin and NLK/β-actin signal ratios normalized with respect to the siGFP-treated group (n = 3 independent experiments). (D) NF-κB transcriptional activity in Kit^+∕+ SMC transduced with lentivirus-encoded siRNAs complementary to TAK1, NLK, or GFP after 24-hour treatment with POVPC (50 µg/ml), as determined by dual-luciferase assay. Transcriptional activity is represented as the mean ± SD of the Firefly/Renilla luciferase ratio normalized with respect to the siGFP-treated group (n = 3 independent experiments). (E) Phosphorylated (pS536) protein levels of NF-κB p65 in POVPC-treated Kit^+∕+ SMC transduced with lentivirus-encoded siRNAs as determined by ELISA. Values are expressed as the mean ± SD of the p-p65/total p65 ratio normalized with respect to the siGFP-treated group (n = 3 independent experiments). (F) Protein expression of the pro-inflammatory mediators MMP-2 and MCP-1 in POVPC-treated Kit^+∕+ SMC transduced with lentivirus-encoded siRNAs as determined by Western blot. Protein expression is expressed as the mean ± SD of the MMP-2/β-actin and MCP-1/β-actin signal ratios normalized with respect to the siGFP-treated group (n = 3 independent experiments). Molecular weight markers are shown on the right side of the gels. ^∗p < 0.05 and ^∗∗p < 0.01 using a one-way ANOVA followed by a Newman-Keuls test.