Dynabeads

Cells were infected with lentiviruses containing pLenti-CMV-puro-3×-FLAG-ΔNLS-DEDD plasmids for 72 h before puromycin (1 μg/mL) treatment for 48 h. The cells were further cultured for two more doublings. The harvested cells were washed with 1× PBS and re-suspended in 1:9 ratio of cell mass to extraction buffer (Dynabeads^® Co-Immunoprecipitation (Co-IP) Kit, Thermo Scientific) with protease inhibitors. The cell lysates were incubated on ice for 30 min and then centrifuged at 2600 × g for 5 min at 4 °C to remove large cell debris and nuclei. BCA (bicinchoninic acid) assay was used to quantify the protein concentration in the supernatant. About 5000 μg of protein was incubated with 1 μg of anti-FLAG antibody (Sigma-Aldrich, F1804)-coupled Dynabeads (10 μg of antibody) on a roller at 4 °C for 1 h. Another 2000 μg of protein was incubated with 1 mg of anti-mouse IgG1 (Cell Signaling, 5415S)-coupled Dynabeads (10 μg of antibody) for non-specific control immunoprecipitation. One percent of cell lysates were saved as input control. The Dynabeads^® Co-IP complexes were washed with extraction buffer for three times. The proteins bound to beads were released in 60 μL of elution buffer provided by the kit. The samples were added five volumes of cold (−20 °C) acetone overnight at −20 °C.

ULI-NChIP was performed for each histone modification mark according to the published method (Brind’Amour et al., 2015 (link)). Briefly, 100,000 cells were FACS-sorted, spun down, and resuspended in nuclear isolation buffer. Chromatin were digested using MNase and immunoprecipitated by incubating with antibody against H3K4me3 (Millipore, 04-745) and H3K27ac (Cell Signaling Technology, 8173) attached protein A:protein G Dynabeads. Next, chromatin were eluted and purified using PCI (phenol:chloroform:isoamyl alcohol). Finally, raw ChIP material was re-purified with 1.8× volume of AMpure XP beads before library construction.

The sequences encoding DMRT2 and FXR were cloned into pcDNA3.1-Flag and pcDNA3.1-HA vectors and named Flag-DMRT2 and FXR-HA, respectively. Then, these vectors were co-transfected into 3T3-L1 adipocytes. The empty vector was co-transfected into target cells as a negative control. After transfection for 48 h, the cell proteins were harvested, and immunoblotting examined the expression of the labeled target protein in cells. For the co-immunoprecipitation assay, cell lysates were centrifuged and the supernatant was incubated with Protein G Dynabeads or Dynabeads conjugated with antibodies against Flag (Sigma-Aldrich) at 4°C overnight. The beads were washed three times with cell lysis buffer and the precipitated proteins were then subjected to electrophoresis and Western blotting for detection of Flag (ab205606, Abcam) and HA (ab9110, Abcam). For determining the endogenous interaction of DMRT2 and FXR, the untransfected 3T3-L1 adipocyte lysate underwent co-immunoprecipitation (co-IP) protocol using Dynabeads conjugated with antibodies against FXR (#72105S, Cell Signaling Tech, USA). The precipitated proteins were then subjected to electrophoresis and Western blotting for detection of DMRT2 and FXR.