Liver tissue lysates obtained from AAV8 control and AAV8 BMP4 mice were precleaned with A/G PLUS-Agarose beads before immunoprecipitation with a PGC1-a antibody (sc-13067, Santa Cruz Biotechnology, Dallas, TX, USA). Samples were then immunoblotted with same PGC1-a antibody or acetylated-lysine antibody (#9681, Cell Signaling). Beads and antibody were used as a negative control.
Acetylated lysine antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Acetylated-lysine antibody is a laboratory reagent used to detect and study acetylated lysine residues in proteins. It is a specific and sensitive tool for identifying and quantifying acetylated proteins in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Hrp igg rabbit antibody, by Jackson ImmunoResearch (2 mentions)
Hrp reacting elisa substrate, by Thermo Fisher Scientific (2 mentions)
Elisa plate, by Thermo Fisher Scientific (2 mentions)
Flag m2 antibody, by Merck Group (2 mentions)
Hdac cell based activity assay kit, by Cayman Chemical (2 mentions)
Mab to p70 s6 kinase, by Cell Signaling Technology (2 mentions)
Streptavidin hrp, by BioLegend (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
G box, by Syngene (2 mentions)
Ecl detection system, by GE Healthcare (2 mentions)
C myc rabbit mab, by Cell Signaling Technology (1 mentions)
Bcl 2 rabbit mab, by Cell Signaling Technology (1 mentions)
Bax rabbit mab, by Cell Signaling Technology (1 mentions)
P53 mouse mab, by Cell Signaling Technology (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
β actin mouse mab, by Cell Signaling Technology (1 mentions)
P tak1, by Cell Signaling Technology (1 mentions)
β mhc sc53090, by Santa Cruz Biotechnology (1 mentions)
Phenylephrine p6126, by Merck Group (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
24 protocols using acetylated lysine antibody
1
Immunoprecipitation of PGC1-α
2
Comprehensive Protein Extraction and Analysis
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Cells were lysed in RIPA buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% NP-40) supplemented with protease inhibitor cocktail. Cell lysates were centrifuged at 12,000 rpm for 30 min at 4 °C, supernatants were saved, and protein concentrations were determined by BCA protein assay (Thermo Scientific, Rockford, Illinois, USA). Equal amounts of total cell lysates or CM of cell culture were subjected to western blot assays as we described previously [41 (link), 42 (link)] to measure protein expression. Antibodies for western blot analyses were from the following sources: NNMT rabbit mAb (E6N2Z, #33361), SIRT1 mouse mAb (1F3, #8469), Hexokinase II rabbit mAb (C64G5, #2867), LDHA rabbit mAb (C4B5, #3582), c-myc rabbit mAb (E5Q6W, #18583), BAX Rabbit mAb (D2E11, #5023), Bim Rabbit mAb (C34C5, #2933), Bcl2 Rabbit mAb (D55G8 #4223), FOXO3a rabbit mAb (D19A7, #12829), p53 Mouse mAb (1C12, #2524), Acetylated-Lysine Antibody (#9441), and β-actin mouse mAb (8H10D10, #3700) (Cell Signaling Technology, Beverly, MA, USA).
3
Acetylation Assay for PGC-1α
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U-2OS human osteosarcoma cells were infected with Flag-tagged-PGC-1α and GCN5 expressing adenovirus and were split to 384 well plates. The chemical compounds were added to the cells and incubated overnight. Cells were harvested and lysates were transferred to ELISA plates (Nunc, 460372) coated with M2 Flag-antibody (Sigma, F1804) in order to immunoprecipitate PGC-1α. The plates were washed with PBST buffer and acetylated-lysine antibody (Cell Signaling, 9441) was added to detect the acetylation level of PGC-1α. After primary antibody incubation, HRP-IgG-Rabbit antibody (Jackson Immuno Research, 711035152) was added as a secondary antibody. A plate reader recorded the signal after incubating the plates with HRP-reacting ELISA substrate (Thermo Scientific, 37069) to generate chemiluminescence at 425nm. Further details for the screening conditions are available upon request.
4
Signaling Pathways in Cardiomyocyte Hypertrophy
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Antibodies against the following proteins were purchased from Cell Signaling Technology (Danvers, MA, USA): GCN5 (3305, 1:1000 dilution), p-TAK1 (9339, 1:1000 dilution), TAK1 (5206, 1:1000 dilution), p-ERK1/2 (4370, 1:1000 dilution), ERK1/2 (4695, 1:1000 dilution), p-JNK (4668, 1:1000 dilution), JNK (9252, 1:1000 dilution), p-p38 (4511, 1:1000 dilution), p38 (9212, 1:1000 dilution), TAB1 (3226, 1:1000 dilution), TAB2 (3,745, 1:1000 dilution), Acetylated-Lysine Antibody (9441 S, 1:1000 dilution) and GAPDH (5174, 1:1000 dilution). Antibodies against ANP (sc20158, 1:200 dilution) and β-MHC (sc53090, 1:200 dilution) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Phenylephrine (P6126) and antibody against α-actinin (A7811, 1:100 dilution) were obtained from Sigma-Aldrich (St. Louis, MO, USA). A BCA protein assay kit, obtained from Thermo Fisher (Waltham, MA, USA), was used to determine the protein concentrations. MB3 (HY-129039), NG25 (HY-15434), and 5Z-7-oxozeaenol (HY-12686) were purchased from MCE (Shanghai, China).
5
HDAC Inhibition Impacts T Cell Activation
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HDAC Cell-Based Activity Assay Kit (Cayman Chemical) was used to measure HDAC activity in activated T cells. Naïve CD4+ T cells, isolated from WT or indicated KO mice, were pre-activated with anti-CD3 and CD28 for 2 days. The activated T cells were incubated with C2, C3, or TSA for 2 hours and processed to measure the HDAC inhibitor activity of the reagents according to the manufacturer’s protocol.
CD4+ T cells were activated with anti-CD3/CD28 and hIL-2 (100 U/ml) in the presence or absence of C2 (10 mM), C3 (1 mM), or TSA (10 nM) for 3 days. Cultured cells (1 × 107 cells) were lysed, pre-cleared by Protein A agarose beads, and incubated with acetylated-lysine antibody (9441, Cell Signaling Technology) overnight at 4°C. Antigen-antibody complexes were precipitated with Protein A agarose beads. Immunoprecipitates or total cell lysate (40 μg of protein/well) were separated with SDS-polyacrylamide gels and then transferred onto nitrocellulose membranes (Bio-Rad). After blocking 1 h in a nonfat dried milk, membranes were incubated with mAb to p70 S6 Kinase (49D7, biotinylated, Cell Signaling Technology) overnight at 4°C. The membranes were developed with HRP-streptavidin (Biolegend) and the ECL detection system (GE Healthcare) using a G:BOX (Syngene).
CD4+ T cells were activated with anti-CD3/CD28 and hIL-2 (100 U/ml) in the presence or absence of C2 (10 mM), C3 (1 mM), or TSA (10 nM) for 3 days. Cultured cells (1 × 107 cells) were lysed, pre-cleared by Protein A agarose beads, and incubated with acetylated-lysine antibody (9441, Cell Signaling Technology) overnight at 4°C. Antigen-antibody complexes were precipitated with Protein A agarose beads. Immunoprecipitates or total cell lysate (40 μg of protein/well) were separated with SDS-polyacrylamide gels and then transferred onto nitrocellulose membranes (Bio-Rad). After blocking 1 h in a nonfat dried milk, membranes were incubated with mAb to p70 S6 Kinase (49D7, biotinylated, Cell Signaling Technology) overnight at 4°C. The membranes were developed with HRP-streptavidin (Biolegend) and the ECL detection system (GE Healthcare) using a G:BOX (Syngene).
6
Acetylation Assay for PGC-1α
7
HDAC Inhibition Impacts T Cell Activation
8
PGC-1α Lysine Acetylation Analysis
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PGC-1α lysine acetylation was analyzed by immunoprecipitation of PGC-1α followed by western blot using acetylated-lysine antibody (1:1000; Cell Signaling, 9681S, MA, USA). Tissue protein extracts and protein content were prepared using RIPA buffer and BCA protein assay kit. For immunoprecipitation, 500 μg of protein lysates samples were incubated at 4°C rotation overnight with anti-PGC1α (2 μg; Santa Cruz, SC-13067, CA, USA) diluted as recommended in the manufacturer's instructions. Purified proteins were separated by 8% SDS-PAGE and immunoblotting using anti-PGC1α (1:200; Santa Cruz, CA, USA) and acetylated-lysine (1:1000; Cell Signaling Technology, USA). The supernatant and precipitated protein was immunoblotted with PGC-1α and actin antibody to confirm the immunoprecipitation process (S2 Fig ).
9
Immunoprecipitation and Western Blot of C1-INH
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The IP of serum C1-INH was performed using a mouse anti-C1-INH monoclonal antibody (M01, Abnova, New Taipei City, Taiwan). Immunoprecipitated C1-INH (100 μg of protein in 8% gel) or serum protein (2 μg of protein in 6% gel) was separated in SDS-PAGE gels and was assessed using Western blotting. Ac modifications of the C1-INH were verified using a rabbit polyclonal acetylated-lysine antibody (#9441 Cell Signaling Technology, Danvers, MA, USA). C1-INH protein levels were evaluated using a mouse anti-C1-INH monoclonal antibody (M01, Abnova). Detailed methods are presented in Supplementary Information .
10
Quantifying Acetylated PGC1α Levels
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Immunoprecipitation was performed as previously described [38 (link)]. To determine the levels of acetylated-PGC1α, the liver nuclear extract from each group was initially immunoprecipitated with anti-PGC1α (Abcam, Cambridge, MA), followed by immunoblotting with acetylated lysine antibody (Cell Signaling Technology, Danvers, MA).
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