Nb100 2289

Western blot analysis was performed as previously described [7 (link)]. We used the following antibodies: for AHR, a rabbit anti-rat AHR antibody (1:1000, overnight incubation; NB100-2289, Novus Biologicals, Littleton, CO, USA); for angiotensin II type 1 receptor (AT1R), a rabbit anti-rat AT1R antibody (1:500, overnight incubation; AB15552, Millipore, Billerica, MA, USA); for angiotensin II type 2 receptor (AT2R), a rabbit anti-rat AT2R antibody (1:250, overnight incubation; sc-9040, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Bands of interest were visualized using enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA, USA) and quantified by densitometry (Quantity One Analysis software; Bio-Rad), as integrated optical density (IOD) after subtraction of background. The IOD was factored for Ponceau red staining to correct any variations in total protein loading. The protein abundance was represented as IOD/PonS.

ChIP assay was performed using a ChIP assay kit (catalog p2078, Beyotime). DNA-bound proteins were fixed using 4% PFA. Chromatin was purified and sonicated to generate fragments of approximate sizes between 500 and 1000 bp. In total, 20 μL of the sonicated chromatin was collected for input. A total of 180 μL of the sonicated chromatin was immunoprecipitated by a rabbit anti-AHR antibody (catalog NB100-2289, Novus, 1:200) and a rabbit nonimmune IgG antibody (catalog AC005, ABclonal, 1:200). The protein-DNA complexes were mixed with 5M NaCl and incubated at 65°C for 4 hours. The purified DNA fraction was used for qPCR analysis. The mouse Cxcl14 promoter primer pair includes the following: forward 5′-TGGACCACGAGCCCAGCAAG-3′, reverse 5′-TTTACTGTCCGAAGCCACCG-3′. The mouse Cxcl14 exon 3 primer pair includes the following: forward 5′-AGAAGATGGTTATCATCACC-3′, reverse 5′-TTCTTCGTAGACCCTGCGCT-3′. Data were normalized with the non–immune rabbit IgG values.

Cell were treated as for qPCR experiments. To prepare protein lysates, cells were harvested, washed, and lysed in ice cold RIPA buffer (150 mM NaCl, 5 mM Tris [pH 8.0], 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with complete protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitors (Roche). Western blotting was performed according to standard protocols. Specific antibodies against AHR (NB100-2289, Novus Biologicals) MDM2 (33–7100, ThermoFisher Scientific), p21 (610233, BD Transduction), p53 (sc-126, Santa Cruz Biotechnology), p53 p-Ser33 (2526 s, Cell Signaling), p53 p-Ser15 (9286 S, Cell Signaling), p53 p-Ser37 (9289 S, Cell Signaling) were used for endogenous protein detection. As loading control, anti-β-actin (MAB1501, Millipore) or anti-GAPDH (sc-365062, Santa Cruz Biotechnology) was used after stripping the membrane with Restore Plus Stripping Buffer (ThermoFisher Scientific). Densitometric analysis of the bands was performed using ImageJ software (http://imagej.nih.gov/ij/). Full-length uncropped blots are displayed in Supplementary Fig. S4.