Detergent compatible assay

Cells were washed twice in phosphate-buffered saline (PBS) and lysed in RIPA buffer for 30 min on ice. After centrifugation at 4°C for 10 min, the supernatant was removed, and the protein concentration was estimated calorimetrically using a Bio-Rad detergent-compatible assay. Protein samples (30 μg) were loaded onto SDS-polyacrylamide gels at a percentage appropriate for electrophoretic separation. Antibodies used for Western blotting were as follows: antibodies against EBNA3A (ab16126; Abcam), EBNA3B (clone 6C9; Allday lab), EBNA3C (clone A10; a gift from M. Rowe, University of Birmingham), EBNA1 (a gift from P. Farrell, Imperial College), EBNA2 (ab90543; Abcam), EBNA-LP (JF-186) (82 (link)), LMP1 (CS1-4; Dako), γ-tubulin (T6557; Sigma), RBPJ (J7A11-161; a gift from B. Kempkes, Helmholtz Zentrum München), KDM2B (09-864; Millipore), BMI1 (05-637; Millipore), SUZ12 (sc-46264; Santa Cruz), and mCherry (Ab183628; Abcam). In all blots, γ-tubulin was used as a loading control. The appropriate horseradish peroxidase (HRP)-conjugated antibodies were used as secondary antibodies (all from GE Healthcare). An ECL kit (Amersham Biosciences) was then used for visualization by autoradiography. In some cases, the membrane used for Western blotting was cut horizontally after protein transfer in order to facilitate multiple antibody probes and a single loading control for each blot used.

Protein extraction of ipsilateral hemisphere of mouse pups was obtained using RIPA lysis buffer (Santa Cruz Biotechnology) with further centrifugation for 30 min at 14,000g at 4 °C. The supernatant was collected, and the protein concentration was determined using a detergent compatible assay (Bio-Rad). Equal amounts of protein were loaded on an SDS-PAGE gel. After being electrophoresed and transferred to a nitrocellulose membrane, the membrane was blocked and incubated with the primary antibody at 4 °C overnight. The primary antibodies included: rabbit anti-TET2 (ABE 364, EMD), rabbit anti-NF-κB p65 (8242, CST), rabbit anti-acetyl-p65 (ab19870, abcam), mouse anti-HDAC3 (3949 s, CST), mouse monoclonal anti-β-actin (A1978, Sigma-Aldrich), or rabbit polyclonal anti-GAPDH antibody (ab9485, Abcam). Nitrocellulose membranes were incubated with secondary antibodies (Santa Cruz Biotechnology) at room temperature for 1 h. Immunoblots were then probed with an ECL Plus chemiluminescence reagent kit (32132, Fisher Scientific) and visualized with the imaging system (Bio-Rad, Versa Doc, model 4000). The images were analyzed using the NIH Image J software.

Cells were plated (500,000 cells per well) and then incubated overnight before harvest. Medium was removed and wells were washed once using 1 mL of PBS per well. Cells were then lysed using 150 µL/2 wells (to increase protein concentration) of the lysis buffer on ice for 10 min, scraped, and collected. Cell lysis buffer preparation: 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 1% Triton; 1 EDTA-free tablet; and 1 PhosSTOP Phosphatase Inhibitor Cocktail Tablet/10 mL of the lysis buffer and then mixed gently for 20 min. Samples were collected in 1.5 mL tubes and centrifuged for 20 min at 11,290× g at 4 °C (Hermle Z 230MR, Labnet International Inc., Cary, NC, USA). The supernatant and pellet were then separated, and both were stored in a −80 °C freezer. A detergent compatible assay (Bio-Rad, Hercules, CA, USA) was used to determine protein concentrations per sample using a BSA standard.

LCLs were harvested and lysed in immunoprecipitation (IP) buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1mM DTT and 0.5% Nonidet P-40) plus protease inhibitors (Roche Molecular Biochemicals). Protein concentration was estimated colorimetrically using the Bio-Rad detergent-compatible assay and 250μg were used per IP. Cell extracts were then pre-cleared with 30μl of Protein G-Sepharose beads (GE Healthcare) at 4°C for 1h. Complexes were precipitated with specific antibodies (S1 Table) and the mixture was incubated at 4°C overnight. Then, 30μl of Protein G-Sepharose beads were added for 1h at 4°C, washed four times in IP buffer and the immunopurified proteins were resolved by SDS-PAGE and detected by western blot.

Protein concentrations in serum and in vaginal canal lysates were determined using enzyme-linked immunosorbent assay (ELISA). IgE concentration was measured using an IgE ELISA kit (Bethyl Laboratories, Montogomery, TX, USA). CXCL2 and IL-1β levels were quantified using cytokine specific ELISA kits (R&D Systems, Minneapolis, MN, USA) from whole cell lysates, as previously described [8 (link),9 (link)]. Absorbances for all ELISAs were measured at 450 nm and 570 nm using a PowerWave XZ microplate spectrophotometer (Biotek Instruments, Winooski, VT, USA). The recorded optical density measurements (OD) were then used to determine the protein concentration for each sample from a standard curve. Total protein concentrations in all samples were determined using a Detergent Compatible Assay (Bio-Rad, Hercules, CA, USA) following the manufacturers’ directions. Total protein concentrations were used to normalize concentrations of target proteins derived from ELISA assays.