Research Article

Hypochlorite-Modified Albumin Upregulates ICAM-1 Expression via a MAPK–NF-κB Signaling Cascade: Protective Effects of Apocynin

Figure 3

HOCl-alb induced the activation of NF-κB in HUVECs. (a) HUVECs were stimulated with 200 mg/L HOCl-alb for 30 min. Translocation of NF-κB/p65 was detected with immunofluorescence. Cells were incubated with rabbit anti-human NF-κB/p65 antibody (10 μg/mL) overnight at 4°C, FITC (green) labeled swine anti-rabbit IgG (1 : 20) at 37°C for 45 min, and 10 μg/mL PI (red) at room temperature in the dark for 10 min and then observed under a confocal microscope at 10 × 40 times magnification. (b) HUVECs were stimulated with 200 mg/L HOCl-alb for the indicated times. NF-κB/p65 was detected in the nuclear extracts (nuc p65) and cytoplasmic extracts (cyt p65) with western blotting. (c) HOCl-alb-induced NF-κB activation in the nuclear extracts was detected with EMSA. Data are expressed as the means ± SD of three independent experiments. ANOVA, in (b); versus 0 min.