Reducing sample buffer

Naïve CD4⁺Foxp3⁻ T cells (WT or Stat6^−/−) were polarized under iTreg-polaring conditions (TGF-β plus IL-2) plus GITR costimulation, and 24 or 48 hours later cells (1 × 10⁷ cells) were collected in IP lysis buffer (87788; Thermo Scientific). Subsequently, cell lysate was incubated with 5µg anti-STAT6 (SC-981X), anti-p50 (sc-1192; both from Santa Cruz Technology) or purified goat IgG (02-6202; Life Technologies) at 4°C overnight and then with Protein G agarose beads (16–201, 40µl; EMD Millipore) for additional 4 hours. The agarose beads were collected by centrifugation and then washed 4 times with IP lysis buffer. Proteins were eluted in reducing sample buffer (39000; Thermo Scientific) and analyzed by immunoblot. FLAG-Foxp3 retrovirus was transduced into naïve CD4⁺Foxp3⁻ T cells (1 × 10⁷ cells) polarized under iTreg-culturing condition with GITR costimulation, and 48 hours later, cells were collected and processed as above except that anti-FLAG antibody (F1804; Sigma-Aldrich) and normal mouse IgG (sc-2025; Santa Cruz Technology) were used for immunoprecipitation.

Cells were washed with phosphate-buffered saline (PBS) and collected using the EpiQuik Total Histone Extraction Kit (EpiGentek). Protein concentrations were determined using the micro BCA kit (Thermo-Fisher Scientific) according to manufacturer’s protocol. Protein samples were supplemented with reducing sample buffer (Thermo Scientific) and denaturized by incubating at 95 °C for 5 min. The nonspecific binding was blocked by incubation of the PVDF membrane in 5% bovine serum albumin (BSA), diluted in Tris Buffered Saline Tween (TBST, 50 mM TRIS, 150 mM NaCl, 0.1% Tween-20 (Applichem) for 1 h at room, temperature followed by incubation with primary antibodies overnight. The antibodies, diluted in TBST, were directed against α-tubulin (1:5000, T6199, Sigma-Aldrich), acetylated α-tubulin (1:5000, T6793 monoclonal, Sigma-Aldrich), histone H3 acetyl k9+k14 (1:1000, 9677L, Cell Signaling), and histone 4 (1:1000, ab10158, Abcam). Secondary antibodies conjugated with horseradish peroxidase (Dako, 1:5000, 1 h, RT) where used prior to detection with ECL substrate (life technologies) with a LAS 4000 Image Analyzer (GE Healthcare). Luminescent signals were quantified with ImageQuant TL 7.0 software (GE Healthcare) and then graphed with GraphPad Prism software. A mild reblotting buffer (Merck Millipore Corp.) was applied to strip the blots.

Naive CD4⁺Foxp3⁻ T cells were transduced with retroviral vector expressing FLAG-RelB, and polarized under Th17 condition for 48 hrs. Cells (15 × 10⁶ cells) were collected in IP lysis buffer (87788; Thermo Scientific). Subsequently, cell lysate was incubated with 10ug anti-FLAG (F1804; Sigma-Aldrich) or control antibody (ab18414; Abcam) at 4° C overnight, followed by incubation with Protein A agarose beads (9863; Cell Signaling Technology) for additional 4hr. Beads were collected by centrifugation and washed 4 times with IP lysis buffer. Proteins were eluted in reducing sample buffer (39000; Thermo Scientific) and analyzed by immunoblot.