Fig 2: Comfrey inhibits NF-?B signaling. (A) Reporter gene assay in HUVEC transfected with an E-selectin promoter-reporter construct (SELE-Luc) and stimulated with IL-1 alone or in the presence of concentrations of comfrey-RE (200, 130, 100 µg/ml; dark bars), comfrey-OP (20, 13, 10 µg/ml; gray bars), or TAK inhibitor (5, 2.5 µM; light bars). Luciferase levels were assayed 16 h later and normalized to the fluorescence from a co-transfected EGFP plasmid used as transfection control. Values are shown as mean fold change in regard to non-stimulated cells. Error bars represent mean ± SD (n = 3). *p < 0.05. (B) Reporter gene analysis as described in A, except that an NF-?B-specific-reporter gene (5 × NF-?B-Luc) was used. (C) Western blot of IL-1ß induced HUVEC (time points as indicated) either left untreated (control, left panels) or preincubated with comfrey-OP (right panels) for 30 min. Samples were analyzed for the presence of I?Ba, phospho-I?Ba (pI?Ba), phospho-IKK1/2 (pIKK1/2), and total IKK2. ß-actin represents the loading control. Note that the slower migrating band seen within the panel depicting I?Ba represents phospho-I?Ba. (D) Western blot as in C, except that the time course was extended to 90 min in order to capture the phase of I?Ba re-synthesis.