Anti gr

ChIP assay was performed by partially modifying the method described previously^{48 (link)}. INS-1D cells were fixed in formaldehyde solution (final concentration, 1%) for 10 min, washed using ice-cold PBS, and collected as before. The cell lysates were sheared by sonication and the DNA was analyzed on a 2% agarose gel. The DNA–protein complexes were immunoprecipitated with 2 μg of anti-GR (#3660, Cell Signaling Technology) or Normal Rabbit IgG (#2729, Cell Signaling Technology). The purified immunoprecipitated DNA was quantified by qPCR using GRE1 specific primers and SYBR green (see Table S8).

Brain tissues were fixed in 4% neutral buffered paraformaldehyde (pH = 7.4) and embedded in paraffin. The paraffin sections (5 μm) were deparaffinized in xylene and rehydrated in ethanol; then, antigen retrieval was performed by incubating in oiled 0.01 M sodium citrate buffer (pH = 6) and blocking endogenous peroxidase activity using 10% H2O2. To reduce nonspecific staining, the slides were immersed in Protein Block, Serum Free (Dako, Tokyo, Japan) for 15 min. For immunohistochemical staining, the sections (5 μm) were incubated with anti-BDNF (1:100, Abcam, Cambridge, UK), anti-GR (1:400, Cell Signaling Technology, Danvers, MA, USA) or anti-doublecortin (1:1000, Abcam, Cambridge, UK) and then incubated with biotinylated goat anti-rabbit IgG and streptavidin peroxidase complex (Nichirei Biosciences Inc., Tokyo, Japan) for 30 min at room temperature, stained with diaminobenzidine and then counterstained with hematoxylin. A light microscope (Olympus, BX50, Tokyo, Japan) connected to a digital camera was used for examining and photographing the slides. For quantification of doublecortin-positive cells in the DG of both hippocampal lobes, the number of immunopositive cells was counted in 10 randomly selected fields of sections, original magnification ×400, as previously described [72 (link)].

The other side of hippocampus was applied for western blotting (Western Blotting Kit, Beyotime Institute of Biotechnology, China). The tissue was homogenized in cell lysis buffer (NP-40 lysis buffer) with protease inhibitor and it was ground by refiner before supernatant was kept in −20°C after centrifuge. The protein was electrophoretically resolved on 10% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes at 100 mA for 2.5 h. After that, the membranes were blocked in skimmed milk for 1 h at room temperature and overnight at 4°C in anti-11β-HSD1 (1 : 1000; Cell Signaling Technology, USA), anti-GR (1 : 1000; Cell Signaling Technology, USA), and anti-β-actin (1 : 800; Cell Signaling Technology, USA), receptively. Then the membrane was incubated with HRP antibody (Boster Co., Wuhan, China) at dilution of 1 : 1000. Finally, the membranes were observed by the use of enhanced chemiluminescence kit (ECL, Amersham).

Primary antibodies: anti-GR (Cell Signaling Technology, #12041), anti-AR (23 (link)), anti-FOXA1 (Abcam, #ab23738), anti-TLE3 (Abcam, #ab94972), anti-H3K27ac (Active Motif, #39133), anti-α tubulin (TUB) (Santa Cruz Biotechnology, #sc-5286). Secondary antibodies: goat anti-rabbit (Invitrogen, #G-21234), goat anti-mouse (Zymed Laboratories, #81–6520), donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher, #A-31573).

Primary antibodies: anti-GR (Cell Signaling Technology, #12,041, RRID:AB_2631286), anti-AR (custom polyclonal rabbit antiserum) [31 (link)], anti-FOXA1 (Abcam, #ab23738, RRID:AB_2104842), anti-EP300 (Santa Cruz Biotechnology, #sc-585, RRID:AB_2231120), anti-H3K27ac (Active Motif, #39,133, RRID:AB_2561016), anti-α tubulin (Santa Cruz Biotechnology, #sc-5286, RRID:AB_628411), anti-H3 (Abcam, #ab1791, RRID:AB_302613). Anti-AR recognize both full length AR and AR-V7 [29 (link)]. Secondary antibodies: goat anti-rabbit (Invitrogen, #G-21234), goat anti-mouse (Zymed Laboratories, #81–6520).