Mm00475162 m1

Murine tissue was stored at −80°C. Total RNA was isolated from the kidneys using TRI Reagent (Sigma-Aldrich) according to a standard protocol. Thereafter, 2 μg of total RNA was reverse transcribed using SuperScript III Transcription Kit (Invitrogen, Carlsbad, CA, USA) and random primers (Invitrogen). Hprt gene was served as the housekeeping reference and was assessed using SYBR Green Master Mix (Invitrogen) and by the following primers: forward 5′ GCT TCC TCA GAC CGG TTT TTG C 3′ and reverse 5′ ATC GCT AAT CAC GAC GCT GGG ACT G 3′. For quantification of Foxp3, Gata3, Rorc, Tnfa, Tbx21, Ccr2, and CCr5, the gene expression assays Mm00475162_m1, Mm00484683_m1, Mm01261022, Mm00443258_m1, Mm00450960, Mm00438270, and Mm01216171 (Applied Biosystems, Foster City, DA, USA) were used, respectively.
Real-Time PCR was performed in duplicates on a CF96 real-time detection system (Bio-Rad, Vienna, Austria). The data was evaluated using the 2-^ΔΔCT method.

RNA isolation from lung macerates of AhR ligand-treated and untreated mice was performed as previously described (37 (link)). A NanoDrop ND-1000 spectrophotometer was used to determine RNA purity and concentration. The cDNA was synthesized using 1 µg of RNA and the high-capacity RNA-to-cDNA kit (Applied Biosystems) according to the manufacturer’s instructions. The cDNA was amplified using TaqMan Universal PCR Master Mix (Applied Biosystems) and pre-developed TaqMan assay primers and probes (Ifng, Mm001168134_m1, Tnf, Mm99999068_m1, Il6, Mm00446190_m1, Il10, Mm00439614_m1, Tgfb1, Mm00117882_m1, Il17, Mm00439618_m1, Il22, Mm01226722_m1, Tbet, Mm00450960_m1; Gata3, Mm00484683_m1; Rorc, Mm01261022_m1; Foxp3, Mm00475162_m1; Gapdh, Mm99999915_g1a, all from Applied Biosystems.). PCR assays were performed on an MxP3000P QPCR System and data were developed using the MxPro qPCR software (Stratagene). The average threshold cycle (CT) values of samples were normalized to the CT value of the Gapdh gene. The relative expression was determined by the 2^-ΔΔCT method.