Benzonaze

IRF3 dimerization was assayed under semi-native conditions. Cells were lysed in ice-cold Pierce RIPA lysis buffer (Thermo Scientific) supplemented with 10 mM NaF, 1x complete protease cocktail inhibitor (Roche) and 5 IU mL⁻¹ benzonaze (Sigma). Protein concentration was determined using a BCA protein assay kit (Thermo Scientific). Whole-cell lysates were mixed with 1x XT Sample Buffer (BioRad); samples were neither reduced nor heated before separation was done on 4–20% Criterion TGX precast gradient gels (BioRad) by SDS-PAGE electrophoresis. Each gel was run initially for 15 min at 70 V and 15 min at 120 V. Transfer onto PVDF membranes (BioRad) was done using a Trans-Blot Turbo Transfer system for 7 min. Membranes were blocked for 1 h with 5% skim-milk (Sigma Aldrich) at room temperature in PBS supplemented with 0.05% Tween-20 (PBST). Membranes were probed overnight at 4 °C with the following specific primary antibody in PBST: anti-IRF3. After three washes in PBST, secondary antibodies, peroxidase-conjugated F(ab)2 donkey anti-rabbit IgG (H + L) (1:10,000) (Jackson Immuno Research) were added to the membrane in PBST 1% milk for 1 h at room temperature. All membranes were washed three times and exposed using either the SuperSignal West Pico PLUS chemiluminescent substrate or the SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific).

786-O cells (5 × 10⁵) were seeded into 6-well plate and treated as desired. Afterwards, each well was pelleted and lysed in 80 μL of ice-cold Pierce RIPA lysis buffer (Thermo Scientific) supplemented with 10 mM NaF, 1× protease inhibitors (Roche), and 5 IU/mL benzonaze (Sigma), respectively. The protein concentration was determined and equilibrated using a Pierce BCA protein assay kit (Thermo Scientific). 25 µg of whole-cell lysates was denatured for 5 min at 95 °C in the presence of 1× XT sample buffer and 1× XT reducing agent (both Bio-Rad). Separation of samples was performed by SDS-PAGE on 4−20% Criterion TGX precast midi protein gels (Bio-Rad) at 75 V until the dye front reached the separating gel and then at 120 V until it reached the bottom of the gel. Precision Plus Dual Color protein ladder (Bio-Rad) was used as separation control. Proteins exceeding a molecular weight of 200 kDa (such as TET2) were separated using 4–8% Criterion XT Tris-acetate precast midi protein gels in the absence of SDS, employing a Tris-acetate buffer system at pH 7.0 (Bio-Rad).

MEFs were trypsinized for 5 min, washed and lysed in a protein extraction buffer (10mM TrisHCl pH7.5, 5mM EDTA, 150mM NaCl, 30mM Na₂HPO₄, 50mM NaF, 10% glycerol, 1% NP40, 1X cOmplete (Roche #11873580001), 1X PhosSTOP (Roche #4906845001), 1/1000 benzonaze (Sigma Cat#E1014)) for 30 min at 4°C. Proteins diluted in Laemmli were resolved on 4–12% Bis-Tris gels (Invitrogen Cat#NP0323BOX) in MOPS buffer (Invitrogen Cat#NP0001) and transferred to nitrocellulose membranes (Bio-Rad Cat#1620112) in a 25mM Tris 200mM glycine 20% ethanol buffer. Blots were blocked in 5% milk in TBS-T (0.1% Tween20 in Tris Base Sodium), incubated with primary antibodies diluted in 3% milk in TBST overnight at 4°C, and incubated with secondary antibodies diluted in 3% milk in TBST for 90 min. Blots were revealed with ECL substrate (Thermo Scientific Cat#32132) and imaged with X-ray films. Primary and secondary antibodies are indicated in S2 Table.