FIG. 2.

AhR expression and function in the CH12.IκBαAA cells. CH12.LX (denoted LX) and CH12.IκBαAA cells (pretreated with [+] or without [−] 100 μM IPTG for 2 h) were incubated for 1 h (A) or 12 h (B) in the absence of further treatment or in the presence of 1 μg/ml LPS with or without a 10 nM TCDD cotreatment. “NA” denotes the unstimulated control. Whole cell protein was isolated and analyzed by Western blot analysis. An anti-AhR antibody identified the AhR protein (approximately 95 kDa) and β-actin served as a loading control. Results are representative of at least 3 separate experiments. C, CH12.LX or CH12.IκBαAA cells were pretreated for 1 h with media alone, dimethyl sulfoxide (DMSO) or the AhR antagonist CH223191 (AhRA, 10 or 30 µM). The cells were then treated with DMSO or 10 nM TCDD and incubated for 8 h. Total RNA was isolated, converted to cDNA, and analyzed by real-time PCR for Cyp1a1 transcripts. Results from 3 to 4 separate RNA isolations per treatment are represented as the relative quantitation (RQ) compared with the respective NA set to 1. The DMSO (0.11% final concentration) vehicle control ranged from less than 1 to 30 RQ and the AhRA alone control ranged from less than 1 to 1.5 RQ (data not shown). Significance between the TCDD+AhRA treatment and the TCDD alone treatment was determined by an unpaired, 2-tailed t test. “**” and “***” denote significance at P < .01 and P < .001, respectively, from the appropriate TCDD treatment. Numbers above bars indicate the percent antagonism induced by the AhRA. AhR, aryl hydrocarbon receptor; CH12.IκBαAA, CH12.LX B-lymphocyte cell line expressing an IPTG-inducible IκBα superrepressor; Cyp1a1, cytochrome P4501a1 gene; IPTG, isopropyl β-d-1-thiogalactopyranoside; LX, CH12.LX parental cells.