Histone 3

Western blot analysis was performed as previously^{4 (link)}. In brief, lung tissue was collected and protein prepared in FastPrep-24 lysing matrix tubes (MP Biomedicals) then lysed using Radio-Immunoprecipitation Assay (RIPA) buffer (50 mM TricCl pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) containing protease (Calbiochem, San Diego, CA, USA) and phosphatase inhibitors (Sigma-Aldrich Corp.). 20 μg protein was run on an SDS 8–12% Tris-Glycine gel (Novex, Life Technologies) and then transferred onto a 0.2-μm nitrocellulose membrane (BioRad) overnight, blocked with 1% milk and probed for GR (1:1,000, clone M-20, sc-1004, Santa Cruz), Cav1 (1:1000, clone N20, Santa Cruz), Cavin (1:1000), actin (1:1000), TFIIB (1:1000, Santa Cruz) used to confirm nuclear lysis, Histone 3 (1:1000, Cell Signalling). Immunoreactivity was visualized using enhanced chemiluminescence (GE Healthcare). Densitometry was performed using ImageJ.

Total protein was collected from freshly dissected rat hearts and cell lysates. The nuclear and cytoplasmic proteins of H9c2 cardiomyoblasts were collected using a nuclear and cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology, Jiangsu, China). Equal amounts of protein were separated on 10% or 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to PVDF membranes (Millipore, Eschborn, Germany). The membranes were blocked for 2 hrs with 5% non‐fat milk at room temperature, then incubated overnight at 4°C with primary antibodies specifically against BRD7 (Sigma‐Aldrich), CHOP (Novus, Littleton, CO, USA), XBP‐1s (Abcam), cleaved caspase‐3, full length caspase‐3, B‐cell lymphoma/leukaemia‐2 (Bcl‐2), Bcl2‐associated X protein (Bax), β‐actin, GAPDH, histone3, phospho‐ERK1/2, total ERK1/2, phospho‐Akt and total‐AKT (all Cell Signaling Technology). After being washed three times, the membranes were incubated with respective secondary antibody for 90 min. at room temperature. Protein contents were visualized using an enhanced chemiluminescent reagent (Bio‐Rad, Hercules, CA, USA).

Cells were lysed in lysis buffer (50 mM Tris/HCl pH 7.4) containing deacylase inhibitors (1 µM trichostatin A and 10 mM NAM) as well as protease inhibitor cocktail (#04693159001, Roche) by sonication on ice, the cell lysate sonication procedure was the same as described in the Desuccinylation activity assays section. Samples were equalized to the same protein level with the lysis buffer following quantification by DC protein assays. Cell lysates (30 µg protein) were separated on NuPAGE 4–12% gradient gels (#NP0322, Thermo Scientific) at 110 V for 35 min, followed by 150 V for 40 min at room temperature. After which, proteins on the gel were immediately transferred to a nitrocellulose membrane at 300 mA for 1 h on ice. Antibodies against SIRT5 (#8782, Cell Signaling Technology), succinyllysine (#401, PTM Biolabs), voltage-dependent anion channel (VDAC) (#ab14734, Abcam) and Histone 3 (#9715, Cell Signaling Technology) were used to identify the respective targets. IRDye Donkey anti-rabbit (#926-32213, LI-COR Biosciences) and Goat anti-mouse (#926-32210, LI-COR Biosciences) were used and signals were detected using an Odyssey scanner (LI-COR). Lane profile analyses was performed using Image J 1.51p.

Whole cell lysates were prepared by lysing cells with RIPA buffer (0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 25 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA) supplemented with a protease inhibitor mixture and PhosSTOP (Roche). Histones were prepared by acid extraction as previously described (Choi et al., 2016 (link)). Proteins were quantified with Bradford assay (Bio-Rad). Equal amounts of proteins were separated with SDS-PAGE and transferred to nitrocellulose membranes. To visualize equal protein loading, blots were stained with Ponceau S. Blots were incubated in 5% non-fat milk in TBST, probed with primary antibodies to HA (1:1,000, Cell Signaling Technology, Cat# 2367), CtBP2 (1:1000, BD Biosciences, Cat# 612044, RRID:AB_399431), Mek1/2 (1:1,000, Cell Signaling Technology, Cat# 9122), alpha-tubulin (Sigma, Cat# T5168), MLL1 (1:1,000, Cell Signaling Technology, Cat# 14197), CtBP1 (1:1,000, BD Biosciences, Cat# 612042, RRID:AB_399431), Myc tag (1:1,000, Cell Signaling Technology, Cat# 2276), V5 (1:1,000, Invitrogen, Cat# R950-25), Histone H3K4me3 (1:1,000, Cell Signaling Technology, Cat# 9751, RRID:AB_2616028), and Histone 3 (1:1,000, Cell Signaling Technology, Cat# 5427) and then were incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized by enhanced chemical luminescence (Cytiva or Pierce).

Total protein was extracted from mouse kidneys and cultured cells using RIPA Lysis Buffer (Beyotime Biotechnology, Nanjing, China). Nucleoproteins were extracted using a commercial kit (Beyotime Biotechnology). Samples (20–40 µg of protein/lane) were separated on 12% (w/v) sodium dodecyl sulfate-polyacrylamide gels; the proteins were electrotransferred to 0.22-µm pore-sized polyvinylidene fluoride membranes (Millipore Sigma, Burlington, MA, USA) and immunoblotted with primary antibodies against Klotho (Abcam; 1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA; 1:200), NF-κB p65 (Cusabio, Wuhan, China; 1:1,000), IκB-α (Abcam; 1:5,000), phosphorylated-IκB-α (Abcam; 1:1,000), COX2 (Proteintech, Rosemont, IL, USA; 1:300), caspase-3 (Proteintech; 1:1,000), Bax (Proteintech; 1:2,000), Bcl2 (Cell Signaling Technology, Danvers, MA, USA; 1:1,000), histone 3 (Cell Signaling Technology; 1:1,000), and β-actin (Servicebio; 1:2,000). They were then incubated with HRP-conjugated secondary antibodies (Proteintech; 1:10,000). The Western blots were developed using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA); band intensities were quantified with the aid of Quantity One ver. 4.6.7 software (Bio-Rad).