Anti rabbit 7074

After 24 h, cells were harvested and dissolved in lysis buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerin, and 200 mM dithiothreitol). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Samples were mixed with Laemmle’s loading buffer and boiled for 7 min. Equal amounts (20–40 µg) of protein extracts were loaded to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12% gel) at 100 mV, followed by blotting onto nitrocellulose membranes for 1 h 50 min at 110 V on ice. After transfer, nitrocellulose membranes stained with Ponceau S. Membranes were blocked for 1 h with 5% nonfat milk (AppliChemGmbH, Darmstadt, Germany, A0830) in tris-buffered saline (TBS) at room temperature, probed overnight with the appropriate primary antibodies (1:3000–1:5000) and followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, anti-mouse 7076S or anti-rabbit 7074S, 1:4000) for 1 h with 2.5% nonfat milk in Tris-buffered saline (TBS) at room temperature. Detection was visualized with ECL (Amersham Biosciences, Piscataway, NJ, USA) and ChemiDoc MP Imaging System (BioRad). The quantitative analysis of Western blot data was accomplished using ImageJ Software (NIH, Bethesda, Rockville, MD, USA).

TMT, ascorbic acid, dimethyl sulfoxide (DMSO), 2′,7′-dichlorofluorescein diacetate (DCF-DA), 2′,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assay kit, superoxide dismutase (SOD) assay kit, and all other chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Phosphoprotein kinase B (p-Akt; Ser 473; 9271S), BAX (2772S), and anti-rabbit (7074S) and anti-mouse (7076S) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Phospho-c-Jun N-terminal kinases (p-JNK; sc-6254), cytochrome c (sc-13560), p-tau (Ser 404; sc-12952), and β-actin (sc-69879) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Cell lysates were resolved on an SDS-PAGE gel followed by immunoblotting with two different antibodies against c-Myc. Recombinant Anti-c-Myc antibody (ab32072, Abcam, 1:1000 dilution) or c-Myc Rabbit mAb (18,583, Cell Signaling Technology, 1:1000 dilution) were used as primary antibodies, and anti-rabbit (7,074 s, Cell Signaling Technology, 1:5000 dilution) was used as the secondary antibody. β-actin Rabbit mAb (8,457 s, Cell Signaling Technology, 1:1000 dilution) was used for loading control.

For western blotting, cells were harvested in lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 30 mM NaF, 5 mM EDTA, 10% (v/v) glycerol, 40 mM β-glycerophosphate, 1% (v/v) Triton X-100, 1 mM sodium orthovanodate, 0.1 mM phenylmethanesulfonylfluoride and 0.07 μM aprotinin. Proteins were separated by SDS–PAGE and transferred to polyvinylidene (PVDF) membranes using semi-dry transfer. Membranes were blocked with 5% (w/v) non-fat milk in PBS containing 0.1% (v/v) Tween-20 (PBS-T) for 1 h before incubation with antibodies. Western blots using antibodies against phosphorylated proteins were performed with TBS containing 0.1% (v/v) Tween-20 and 5% bovine serum albumin (BSA).
The antibodies used were anti-IFI16 (1G7, Santa Cruz Biotechnology), anti-cGAS (HPA031700, Sigma Aldrich), anti-STING (D2P2F, Cell Signaling Technology), anti-TBK1 (D1B4, Cell Signaling Technology), anti-IRF3 (D6I4C, Cell Signaling Technology), anti-β-actin (A2228, Sigma Aldrich), anti-phospho(Ser172)-TBK1 (D52C2, Cell Signaling Technology) and anti-phospho(Ser396)-IRF3 (4D4G, Cell Signaling Technology). Primary antibodies were used at a dilution of 1:1,000. Secondary horse radish peroxidase-coupled anti-mouse (7076 S) and anti-rabbit (7074 S) antibodies were from Cell Signaling Technology and used at a dilution of 1:3,000. Full immunoblots including size markers are shown in Supplementary Fig. 6.

Total liver tissue lysates were fractioned by 10% acrylamide gel. The proteins were then transferred to a nitrocellulose membrane and blocked in 5% milk protein diluted in TBST (0.1%) for 1 h. All primary Ab incubations were overnight: NF-κB p65 (8242s; Cell Signaling Technology), acetyl–NF-κB p65 (Lys³¹⁰) (3045s; Cell Signaling Technology), β-actin (A5441; Sigma-Aldrich), acetyllysine (ab80178; Abcam), IKBα (4814S; Cell Signaling Technology), Sirt1 (07-131; MilliporeSigma), PPAR γ coactivator-1a1 (PGC-1α; NBP1-04676; Novus Biologicals), and PBEF (nicotinamide phosphoribosyltransferase [NAMPT]) (sc-393444; Santa Cruz Biotechnology). Next day, membranes were washed in TBST and incubated with secondary Abs (anti-mouse (A4416; Sigma-Aldrich) and anti-rabbit (7074S; Cell Signaling Technology) for 1 h. Proteins were detected with chemiluminescence (Amersham Bioscience).