Ptm 401

Mouse lung tissue samples were lysed in RIPA buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol) with protease/phosphatase inhibitor cocktail (5872S, Cell Signaling Technology, 1:1000), 0.3 mM DTT, 5 mM nicotinamide, and 1 mM sodium butyrate. Total 500 μg of protein were used for immunoprecipitation with indicated dilution of each antibody (anti-STAT1, 14994, Cell Signaling Technology, 1:100).
Western blotting was performed according to standard protocols. The antibodies and dilutions were anti-β-actin (sc-5286, Santa Cruz, 1:10,000), anti-STAT1 (14994, Cell Signaling Technology, 1:1000), anti-acetyl-lysine (PTM-105, PTM Bio, 1:1000), and anti-succinyl-lysine (PTM-401, PTM Bio, 1:1000).

The tissue homogenates were diluted in Laemmli buffer and subjected to SDS-PAGE as described previously [44 (link)]. The levels of total protein succinylation and glutarylation were estimated by Western blotting using primary antibodies #PTM-401 and #PTM-1151, respectively, from PTM Biolabs (Chicago, IL, USA). The levels of total protein acetylation, sirtuin 3, sirtuin 5, PDHA1 and phosphorylated PDHA1 were estimated using antibodies #9841, #5490, #8782, #3205 and #37115, respectively, from Cell Signaling Technology (Danvers, MA, USA). All primary antibodies were used in a 1:2000 dilution. Secondary HRP-conjugated anti-rabbit antibody from Cell Signaling Technology, #7074 (Danvers, MA, USA) was used in 1:3000 dilution. Relative chemiluminescence was detected using ChemiDoc MP Imager (Bio-Rad, Hercules, CA, USA) and processed in Image Lab software v. 6.0.1 (Bio-Rad, Hercules, CA, USA). Normalization was done per total protein level in gel lane, determined via 2,2,2-tricholoroethanol staining as described elsewhere [48 (link)]. When comparing several membranes, the band intensities from different membranes were further normalized to the levels in common samples present in all the membranes. Raw images of membranes with visualized protein bands are presented in Supplementary Figure S1.

Western blotting was performed according to standard procedures. Antibodies used were anti-SIRT7 (Santa Cruz Biotechnology, sc-365344, 1:500), anti-HA (MBL, M180-3, 1:2,000), anti-Flag (Sigma, F3165, 1:10,000), anti-β-actin (Sigma, A1978, 1:10,000), anti-tubulin (Sigma, clone B-5-1-2, T6074, 1:50,000), anti-SIRT6 (Abgent, AP-6245a, 1:500), anti-PARP1/2 (Santa Cruz Biotechnology, sc-7150, 1:5,000), anti-Ku80 (Santa Cruz Biotechnology, sc-5280, 1:2,000), anti-BRCA1 (Proteintech, 22362-1-AP, 1:1,000), anti-γH2AX (Millipore, 05-636, 1:2,000), anti-H2AX (Abcam, ab11175, 1:2,000) anti-pan-acetylation (PTM BioLabs, PTM-105, 1:1,000), anti-pan-succinylation (PTM BioLabs, PTM-401, 1:1,000), anti-H3K122succ (1:4,000), anti-H2BK120succ (1:8,000), anti-H3K122ac (Abcam, ab33309, 1:2,000), anti-H3K18ac (PTM BioLabs, PTM-114, 1:1,000), anti-H3 (Abcam, ab1791, 1:100,000) and anti-rabbit (Jackson ImmunoResearch, 115-035-003, 1:8,000) or anti-mouse (Jackson ImmunoResearch, 111-035-003, 1:8,000) secondary antibodies conjugated to horseradish peroxidase. The bands were quantified by densitometry with ImageJ software. Uncropped scans of the most important blots are shown in Supplementary Fig. 11.

The cell lysates were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (ISEQ00010, Millipore). Subsequently, the membranes were blocked with 5% skimmed milk and incubated overnight at 4 °C with primary antibodies. The following antibodies were used: anti-SIRT5 (1:1000 dilution; 8782 S, Cell Signaling Technology, Danvers, MA, USA), anti-HINT1 (1:1000 dilution; 67583-1-Ig, Proteintech, Rosemont, IL, USA), anti-Succinyllysine (1:1000 dilution; PTM-401, PTM Bio, Hangzhou, China), anti-Acetyllysine (1:500 dilution; PTM-105RM, PTM Bio), anti-GFP (1:5000 dilution; 50430-2-AP, Proteintech), anti-Flag (1:5000 dilution; 80010-1-RR, Proteintech), and anti-β-Actin (1:5000 dilution; AB0035, Abways, Shanghai, China). Then PVDF membranes were incubated with HRP-conjugated goat anti-rabbit or mouse antibodies (1:5000 dilution; AB0101 and AB0102, Abways) and thoroughly washed. The protein bands were visualized using the ECL chemiluminescence detection kit (WBKLS0500, Millipore), and band intensity was quantified using ImageJ software.