Anti ahr

For AHR protein detection in cell culture experiments, cells were lysed in RIPA buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 µg/ml leupeptin). For detection of hepatic AHR, frozen liver tissue (50–80 mg) was homogenized in 400 µl RIPA buffer. The homogenate was sonicated with Bioruptor on the low setting at 4°C for 5 min, 30 s on, and 30 s off. The samples were then centrifuged at 20 000 × g for 10 min at 4°C. Twenty micrograms of protein were separated by SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked for 2 h at room temperature (RT) in 5% nonfat milk dissolved in Tris-buffered saline (TBS)-0.1% Tween20. Membranes were then incubated overnight at 4°C with anti-AHR (1:10 000; Enzo Life Sciences SA210; lot no. 04011942) followed by incubation with anti-rabbit IgG antibody; 1:6250; 1 h incubation RT. PVDF membranes were stripped and incubated with anti-β-actin antibody 1:4000 (Sigma-Aldrich; A-2228) followed by incubation with anti-mouse IgG antibody for 1 h at RT. After membranes were washed with TBST, SuperSignal West Dura (ThermoScientific) was used for detection. The pixel density of protein bands was analyzed with the ImageJ software (imagej.nih.gov/ij). The corresponding βACTIN band intensity normalized AHR signal.

Cells from AHR^Vav1 tissues were washed twice with PBS, and lysed (Reporter Lysis Buffer, Promega, Madison, WI). Lysates were stored at −80°C. Lysate proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) (8% acrylamide resolving gel) and transferred to a polyvinylidene fluoride (PVDF) membrane, which was blocked using 5% nonfat milk in wash buffer (50mM Tris base, 150mM NaCl, and 0.2% Tween 20, pH 7.5). Antibodies used were anti-AHR (rabbit polyclonal, Enzo Life Sciences, Farmingdale NY) and anti-β-actin (rabbit polyclonal, Sigma, St Louis, MO). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson Immunoresearch (West Grove, PA). Proteins were visualized by chemiluminescence using LumiGlo reagents (KPL, Gaithersburg, MD).

5x10⁶ cells CD19⁺CD21^hiCD24^hi and FO B cells were FACS sorted from arthritic WT mice and lysed for 15 minutes at 4°C with cell lysis buffer (Cell signaling technology) for extraction of whole cell lysate. Protein was resolved by SDS-PAGE, transferred to nitrocellulose membranes and blotted using anti-AhR at 1/1000 (Enzo Life sciences) and anti-β-actin at 1/1000 (Cell Signaling Technology). Bound antibodies were revealed with a goat-anti rabbit H&L HRP-conjugated secondary antibody (1/1000) and ECL western blotting substrate (ThermoFisher Scientific).

SDS–PAGE and immunoblotting were performed using total protein extracts obtained from AhR+/+ and AhR−/− mouse livers as described [13 (link)]. Briefly, tissues were extracted and homogenized in ice-cold lysis buffer; following centrifugation, protein concentration was determined in the supernatants using the Coomassie Plus protein assay reagent (Pierce) and bovine serum albumin (BSA) as a standard. Aliquots of 20–30 μg protein were electrophoresed in 8% SDS-PAGE gels, which were transferred to nitrocellulose membranes. After blocking in a TBS-T (50 mM Tris-HC1 pH 7.5, 10 mM NaCl, 0.5% Tween 20) containing 5% non-fat milk, blots were sequentially incubated with the primary antibodies anti-AhR (Enzo 1:1000), anti-p53 (Cell signaling 1:1000) and anti-β-Actin (Sigma 1:1000) and secondary antibodies, washed in TBS-T and developed using the Super-signal luminol substrate (Pierce). Blots were scanned and protein expression quantified using ChemiDoc XRS+ equipment (Bio-Rad).