Amersham biosciences

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).

Cells were grown in 10-cm dishes, exposed to ligands, and then lysed as previously described.^{57 (link)} Equal amounts of whole-protein extract were resolved on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Biosciences, Sigma-Adrich, Milan, Italy), which were probed with primary antibodies against pEGFR Tyr 1173, EGFR (1005), phosphorylated ERK1/2 (E-4), ERK2 (C-14), p-c-Jun S73, c-Jun (N), c-fos (E8), p53 (DO-1), p21 (H164) and β-actin (C2) (Santa Cruz Biotechnology) and then revealed using the chemiluminescent substrate for western blotting Westar Nova 2.0 (Cyanagen, Biogenerica, Catania, Italy).