Western Blotting, Succinylated-Wheat Germ Agglutinin Lectin Precipitation, and IP

For Western blotting assays, cells were lysed with buffer A (150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH7.4), 1% NP-40) supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany) and a phosphatase inhibitor cocktail (Roche, Germany). Preparation of sWGA was as previously described (20). Briefly, 1.5 to 2 mg of total cell lysates were incubated with agarose-conjugated sWGA (Vector Laboratories, Burlingame, CA, USA) overnight at 4°C. For IP, 1.5 to 2 mg of total cell lysates were incubated with agarose-conjugated anti-FLAG antibody (MBL, Woburn, USA) or anti-Myc antibody (MBL, Woburn, USA) for 2 h at 4°C. For IP of endogenous MAVS, 3 mg of total cell lysates were incubated with anti-MAVS antibody (#166583, Santa Cruz, Dallas, TX, USA) overnight at 4°C followed by agarose-conjugated protein A/G (Santa Cruz, Dallas, TX, USA) for 2 h at room temperature. Purified proteins in sWGA/IP precipitates were washed four times with buffer B (150 mM NaCl, 2 mM EGTA, 2 mM MgCl₂, 20 mM HEPES (pH 7.4), and 0.1% NP-40) and were eluted with sodium-dodecyl sulfate (SDS) loading buffer at 95°C for 5 min. The eluents were analyzed via Western blots probed with specific antibodies described in the section to follow. For co-IP experiments, 1.5 to 2 mg of total cell lysates were lysed with buffer C (150 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 50 mM Tris-HCl (pH 7.4), and 0.5% Triton-X100). For Western blotting, 20 to 30 μg of total cell lysate was loaded onto 8% to 10% SDS-PAGE gel. Exceptionally, 60 μg of total cell lysate was loaded on the SDS-PAGE gel to detect p-IRF3. The same amount of protein was loaded in each experiment. After separation onto SDS-PAGE gel, proteins are transferred to NC membrane to detect signals. EZ-Western kit (DoGenBio) or SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.) and Amersham Imager 600 (GE Healthcare Life Sciences, Little Chalfont, UK) were used to signal detection. For quantifying signals, the immunoreactive protein band was detected and the integrated signal intensity was measured using AI600 imager system software. Thereafter, O-GlcNAcylation levels were normalized to integrated signal intensity of β-actin or GAPDH, the loading control of the same gel for each cell type. OGT protein levels were normalized to β-actin, a loading control of the same gel. To quantify proteins and O-GlcNAcylation levels, Loading control proteins including β-Actin and GAPDH, and target proteins to observe or O-GlcNAc levels were measured using AI600 imager system (GE Healthcare, Chicago, IL, USA) software.