The inhibitory effect of pinocembrin on TGF-β1-induced NF-κB transcriptional activity/expressions of NF-κB/IκBα phosphorylation and degradation in Y-79 cells. (A) Cells were pretreated with TGF-β1 (10 ng/ml) for 2 h and then treated with 5 μ M pinocembrin for 0, 1, 2, 3, and 9 h, and the NF-κB transcriptional activity by luciferase report gene assays, as described in Methods. (B) Nuclear extracts were subjected to SDS–PAGE followed by Western blotting with specific antobodies (anti-NF-κB p50, anti-NF-κB p65, anti-p-IκBα, anti-IκBα). C23 and β-actin were used as internal control. (C) NF-κB DNA-binding activity by EMSA, as described in Methods. Lane 1: nuclear extracts incubated with 100-fold excess unlabeled consensus oligonucleotide (comp.) to confirm the binding specificity. Lane 2 represents nuclear extract from Y-79 cells in the absence of TGF-β1 (negative control). Values represent mean ± SD of three independent experiments. (*p < 0.05, **p < 0.01, ***p < 0.001 compared with the TGF-β1 treatment only).