Sc 393232

Cos-7 cells were seeded onto 10-cm² dishes the day before transfection. The following plasmids psg5-V5-PRMT5, pcdna3.1-HA-HP1γ, and pcdna3.1-GR were transfected into Cos-7 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. 48 h after transfection, cells were treated (or not) with Dex for 24 h, and cell extracts were prepared in RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 0.25% deoxycholate) supplemented with protease inhibitor tablets and phosphatase inhibitors (1 mM NaF, 1 mM Na₃VO₄, and 1 mM β-glycerophosphate). Protein extracts were incubated with HP1γ primary antibody (ab10480; Abcam), PRMT5 (ab109451; Abcam) or GR/DGH2L (#12041; Cell signaling) over night at 4°C under agitation. Protein A Agarose (Millipore) beads were then added, and the mixture was incubated 2 h at 4°C. The immunoprecipitates were separated on SDS–PAGE. Immunoblotting was conducted with primary antibodies against GR G-5 (sc-393232; Santa Cruz), GR/D6H2L (#12041; Cell signaling), HP1γ (ab10480; Abcam), and PRMT5 (07-405; Millipore). Secondary antibodies were used for chemiluminescence detection using the ECL detection reagent (Roche Molecular Biochemicals) according to the manufacturer’s instructions. For immunoprecipitation experiments, 3% of the input of each sample was analyzed by immunoblot.

Total protein extracts with RIPA lysis buffer (Beyotime Biotechnology, Jiangsu, China) and protein quantities were measured by the BCA Protein Assay kit (Beyotime Biotechnology, Jiangsu, China). Antibodies against GRα (sc-393232, Santa Cruz, USA) and tubulin (sc-5274, Santa Cruz, USA) was used in Western blot analysis. Images were captured with VersaDoc 4000MP system (Bio-Rad).

Guinea pig antibody to Hap1 (EM77) has been described previously (Li et al., 1996 (link), 2000 (link)). We also used the following antibodies: mouse monoclonal antibodies to GR [sc-393232; Santa Cruz Biotechnology (Santa Cruz, CA, USA)] and vinculin [MAB3574; MED millipore (Billerica, MA, USA)]. Secondary antibodies were horseradish peroxidase (HRP)-labeled donkey anti-mouse, donkey anti-guinea pig, donkey anti-mouse Alexa Fluor 488 or 594, and donkey anti-guinea pig Alexa Fluor488 or 594 from Jackson ImmunoResearch (West Grove, PA, USA). Tamoxifen (TM), Dex, and cycloheximide (CHX) were purchased from Sigma (St. Louis, MO, USA). Guide RNA (gRNA) backbone vector and Cas9 plasmid were purchased from Addgene (Cambridge, MA, USA).

Details of the specific antibodies that were used in the study are as follows: anti-CEP128 (HPA001116, Sigma-Aldrich, WB: 1:1000, IF: 1:200); anti-Ac-Tubulin (ab24610, abcam, IF: 1:1000); anti-NIN (A8215, ABclonal, WB: 1:1000); anti-CEP170 (27325-1-AP, Proteintch, WB: 1:500); anti-YY1 (sc-7341, Santa Cruz Biotechnology, WB: 1:500); anti-GR-β (sc-393232, Santa Cruz Biotechnology, WB: 1:500); anti-SEPT4 (Septin 4) (A10238, ABclonal, IF: 1:100); anti-RCBTB2 (13225-1-AP, Proteintech, WB: 1:1000, IF: 1:50); anti-WBP2NL (22587-1-AP, Proteintech, WB: 1:500, IF: 1:50); anti-CRISP1(MAB4675, R&D Systems, WB: 1:500, IF: 1:50); anti-PRSS55 (bs-19443R, Bioss, WB: 1:1000, IF: 1:50); anti-ubiquitin (ab7780, Abcam, WB: 1:500), anti-GAPDH (ab8245, Abcam, WB: 1:1000); anti-CEBPβ (sc-7962, Santa Cruz Biotechnology, WB: 1:500); anti-Centriolin (sc-365521, Santa Cruz Biotechnology, WB: 1:500).

Cells were lysed in E1A buffer (50 mM HEPES pH7.6, 250 mM NaCl, 5 mM EDTA, and 0.5% NP-40) by incubating for 10 min on ice. After centrifuging, the supernatant was collected in new Eppendorf tubes, and protein concentrations were measured using Bradford. Protein samples containing 50 μg protein were separated by electrophoresis in a 10% gradient SDS polyacrylamide gel and transferred to nitrocellulose membranes (pore size 0.45 μm). After blocking the membranes with 1:2 dilution of Starting Block/PBT (Thermo Fisher Scientific), membranes were incubated overnight at 4 °C with primary antibody to detect GR (G-5) (sc-393232, Santa-Cruz, Bio-connect) or β-actin (MA5-15739, Pierce). Blots were washed with PBT and then incubated for 1 h at room temperature with secondary antibody, anti-mouse antibody (926–32,220; Li-Cor), and immunoreactive bands were detected using ECL (Amersham).

Immunofluorescence analyses were performed using a modified method described by the previous studies (Zhang et al., 2016 (link); Zhao et al., 2017 (link)) to observe the 5-HT and GR relative intensity in dorsal raphe nuclei (DRN) and the hippocampal CA1 area, respectively. Brain tissues were cryoprotected in 30% sucrose, embedded in tissue-freezing medium with liquid nitrogen, and cut into frozen coronal sections (35 μm) using a Leica CM3050 cryostat. Sections were stored under anti-freeze buffer. Parallel free-floating sections were treated with blocking buffer (5% normal chicken serum in PBS and 0.3% Triton X-100 for 1 h at 4°C) and incubated with primary antibodies against 5-HT (1:400, ab66047, Abcam) or GR (1:200, sc-393232, Santa Cruz) overnight at 4°C. After washing with ice-cold PBS, sections were incubated with donkey anti-goat IgG H&L (1:400, Alexa Fluor^® 488, ab150129, Abcam) or goat anti-mouse IgG H&L (1:400, Alexa Fluor^® 594, ab150116, Abcam) secondary antibodies for 2 h at 4°C. The sections were subsequently exposed to DAPI (1:1,000, D9542, Sigma) to stain cell nuclei. All immunoreactions were observed under an Axiophot microscope (Carl Zeiss, Germany). Total signal intensity was quantified using the ImageJ 1.46 version (NIH, Bethesda, MD, USA) and compared relatively with the control group.