Figure 3.

ArCC treatment significantly inhibited TNF-α-induced nuclear factor-κB (NF-κB) activity with translocation of NF-κB from the nucleus to the cytosol. (A) Transcriptional activity of NF-κB in TNF-α-stimulated HUVECs following treatment with 100 or 300 μg/mL of ArCC for 6 h as determined by luciferase reporter gene assay. Data are mean ± SE (n = 3). * Significantly different (p < 0.05) compared with TNF-α-only, ^# p < 0.05 compared with control by one-way ANOVA followed by Bonferroni’s test. (B) Immunoblotting for p65 NF-κB using nuclear fraction prepared from TNF-α-stimulated HUVECs following treatment with 100 or 300 μg/mL of ArCC for 6 h. The blot was stripped and re-probed with anti-poly (ADP-ribose) polymerase (PARP) antibody and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to ensure equal protein loading without cross-contamination of the nuclear and cytoplasmic fractions. (C) Immunocytochemical analysis for p65 NF-κB localization in TNF-α-stimulated HUVECs following 6-h of treatment with 300 μg/mL of ArCC. Staining for p65 NF-κB and DNA is indicated by green and red fluorescence, respectively. p65 NF-κB localization was observed using a fluorescence microscope at 100× magnification