Normal igg

ChIP-DNA prostate tissues of hHGFtg:H11^hMET/+:PB^Cre4 and hHGFtg:H11^hMET/+:Ctnnb1^L(Ex3)/+:PB^Cre4 mice was obtained by ChIP assay. Briefly, mouse prostate tissues were minced, cross-linked with 1% formaldehyde for 25 min at room temperature (RT), and quenched with 150 mM glycine for 10 min. Samples were homogenized in cell lysis buffer (140 mM NaCl, 50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 10% Glycerol, 0.5% NP-40, and 0.25% Triton X-100). The chromatin was resuspended in nuclear lysis buffer (0.2% SDS, 10 mM Tris-HCl [pH 8.0], 1 mM EDTA, and 0.5 mM EGTA) and fragmented to an average size of 200–500 bp with a Sonic Dismembrator Model 100 (Thermo Fisher Scientific) at 4 °C. After centrifugation, the cell sonicate was diluted with ChIP dilution buffer (0.01% SDS, 167 mM NaCl, 16.7 mM Tris-HCl [pH 8.1], 1.1% Triton X-100, and 1.2 mM EDTA), pre-cleared using Dynabead Protein G (10003D, Invitrogen) and then was subjected to immunoprecipitation with Dynabead Protein G conjugated with SP1 antibody (Novus, NB600-233) or normal IgG (Cell Signaling) for 4 h at 4 °C. Crosslinks were reversed, and then chromatin DNA fragments were analyzed by qRT-PCR with specific primers (Supplementary Table 1).

Lysates containing 4 mg of total protein were made using NP40 lysis buffer (Promotor, Wuhan, China) in TKPTS cells cultured on plates and were incubated with 4 μg of an anti-β-arrestin2 antibody at 4°C overnight. Normal IgG (2729S, Cell Signaling Technology) was used as a negative control. Then Protein A/G Plus Agarose (sc-2003, Santa Cruz Biotechnology) was put into the compound for 8 hours at 4°C. After the beads were washed 3 times, the lysates were resuspended and boiled. Immunoblotting was operated as a method of Western blot.

Fluocinolone acetonide (FA), E₂, DHT, RU0486 (Mifepristone), and ICI182,780 (Fulvestrant) were from Sigma-Aldrich (St. Louis, MO, USA). Croton oil (CO) was from Santa Cruz Biotechnology (Dallas, TX, USA). We used antibodies against GR (Santa Cruz Biotechnology), ER (EMD Millipore, Danvers MA, USA), REDD1 (Proteintech Group, Rosemont, IL, USA), GAPDH (Sigma-Aldrich), tubulin, and normal IgG (Cell Signaling, San Jose, CA, USA).