Ahr d5s6h

Cells were grown in 10-cm dishes, exposed to treatments and then lysed in 500 μL of 50 mmol/L NaCl, 1.5 mmol/L MgCl₂, 1 mmol/L EGTA, 10% glycerol, 1% Triton X-100, 1% sodium dodecyl sulfate (SDS), and a mixture of protease inhibitors containing 1 mmol/L aprotinin, 20 mmol/L phenylmethylsulfonyl fluoride and 200 mmol/L sodium orthovanadate. Protein concentration was determined using Coomassie (Bradford) protein reagent according to the manufacturer’s recommendations (Life Technologies, Milan, Italy). Equal amounts of whole protein extract were resolved on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham Biosciences, Sigma-Aldrich, Milan, Italy), probed overnight at 4 °C with antibodies against CYP1B1 (TA339934) and cyclin D1 (TA801655) (purchased from OriGene Technologies, DBA, Milan, Italy), GPER (AB137479) (Abcam, Euroclone, Milan, Italy), AHR (D5S6H) (Cell Signalling technology, Euroclone, Milan, Italy), c-Fos (E8), pEGFR Tyr 1173 (sc-12,351), EGFR (1005), phosphorylated ERK1/2 (E-4), ERK2 (C-14) and β-actin (C-2) (purchased from Santa Cruz Biotechnology, DBA, Milan, Italy). Proteins were detected by horseradish peroxidase-linked secondary antibodies (Bio-Rad, Milan, Italy) and then revealed using the chemiluminescent substrate for western blotting Westar Nova 2.0 (Cyanagen, Biogenerica, Catania, Italy).

CH-223191 was purchased from Selleck. L-kynurenine and BAY11-7085 were purchased from MedChemExpress. Antibodies were used as following: AHR (D5S6H; Cell Signaling Technology), RelA (D14E12; Cell Signaling Technology), IκBα (L35A5; Cell Signaling Technology), phospho-IκBα (14D4; Cell Signaling Technology), α-Tubulin (DM1A; Cell Signaling Technology), LaminB1 (D9V6H; Cell Signaling Technology), HTLV-1 Tax (1A3; Abcam), HTLV-1 gp46 (67/5.5.13.1; Abcam), HTLV-1 p24 (46/3.24.4; Abcam), HTLV-1 p19 (TP-7; Abcam), β-Actin (AF0003; Beyotime Biotechnology), GAPDH (AF0006; Beyotime Biotechnology).