Anti acetyl lysine

The primary antibodies included anti-vWF (Santa Cruz, #sc-365712), anti-COL 1A1 (Proteintech, #14695-1-AP), anti-progerin (Santa Cruz, #sc-81611), anti-Lamin A/C (Cell Signaling Technology, #4777S), anti-Lamin B1 (Proteintech, #66095-1-Ig), anti-SIRT1 (Abcam, #ab110304), anti-LC3B (Abcam, #ab48394), anti-acetyl Lysine (Abcam, #ab22550), anti-Caveolin-1 (Cav-1) (Abcam, #ab32577), anti-Histone H3 (Proteintech, #17168-1-AP), and anti-GAPDH (Proteintech, #60004-1). The secondary antibodies included Cy3-labeled goat anti-mouse IgG (H+L) (Beyotime, #a0521), FITC-labeled goat anti-rabbit IgG (H+L) (Beyotime, #a0562), HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L) (Proteintech, #SA00001-1), and HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (Proteintech, #SA00001-2).
The reagents used included carbon tetrachloride (CCl₄) (Sigma-Aldrich, #56-23-5), hydrogen peroxide (H₂O₂) (Sigma-Aldrich, #1.07298), N-Acetylcysteine (NAC) (MedChemExpress, #616-91-1), mitochondria 2,2,6,6-tetramethylpiperidinooxy (mito-TEMPO) (MedChemExpress, #1334850-99-5), 3-Methyladenine (3-MA) (Sigma-Aldrich, #S2767), rapamycin (sirolimus) (Sigma-Aldrich, #S1039), MCDB131 (Gibco, #10372019), 1640 (Gibco, #11875101), and fetal bovine serum (FBS) (Biological Industries, #04-007-1A).

Proteins from cultured PTCs or kidneys or mitochondria were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes. The blots were incubated with anti-SIRT3 (cat: 5490, CST), anti-Acetyl-lysine (cat: ab22550, Abcam, Cambridge, UK), anti-E-Cadherin (cat: 610181, BD Biosciences, San Jose, CA, USA), anti-Na/K-ATPase (cat: 3010, CST; ab76020, Abcam), anti-AQP1 (cat: AB2219, Millipore, Billerica, MA, USA), anti-FN (cat: F3648, Sigma Aldrich, St. Louis, MO, USA), anti-Collagen I (cat: 1310-01, Southern Biotech, Birmingham, AL, USA), anti-PDH-E1α (cat: ab110334, Abcam), anti-p-PDH (Ser293)(cat: ab92696, Abcam), anti-CPT1a (cat: ab128568, Abcam), anti-ATP5O (cat: ab110276, Abcam), anti-α-tubulin (cat: T9026, Sigma Aldrich), and anti-SDHA (cat: 11998, CST). Quantification was performed by measuring the intensity of the signals with the aid of the National Institutes of Health Image J software package.

Immunoblotting was performed according to methods previously studied in our laboratory [23 (link)]. Protein was first extracted from adipose tissue and cells using lysis buffer (Solarbio, Beijing, China). Approximately 30 μg of protein was separated by electrophoresis (12% and 15% SDS-PAGE gels) and then transferred to PVDF nitrocellulose membranes (Millipore, MA, USA), which were then blocked by 5% skim milk for 2 h. After blocking, these membranes were incubated overnight with various primary antibodies, including anti-PPARG, anti-FABP4, anti-c/EBP α, anti-ATGL, anti-Succinyllysine, anti-Acetyl Lysine, and anti-β-Actin (ABCAM, Cambridge, UK). The membranes were then washed and incubated with HRP-conjugated secondary antibodies for 2 h. Proteins were imaged using chemiluminescent Peroxidase substrates (Millipore) and quantified using a ChemiDoc XRS system (Bio-Rad, Richmond, CA, USA).

PAX3 proteins were immunoprecipitated from the total nuclear protein fraction with either rabbit polyclonal (Abcam, 2.5μg/ml), or rabbit monoclonal (Life Technologies, clone 16H22L10) antibodies. The antibody/antigen complex was immunoprecipitated using anti-IgG agarose beads (Rockland), or Pierce Direct IP Kit (Pierce Biotechnology). Both direct and indirect methods of immunoprecipitation were performed to optimise the results. Precipitated proteins were run on a NuPAGE Bis-Tris precast gel. The following antibodies were used for western blotting: anti-PAX3 (mouse monoclonal, DSHB, 1/1000; or rabbit monoclonal, Life Technologies, clone 16H22L10), anti-phosphoserine (rabbit polyclonal, Abcam, 2.5μg/ml), anti-acetyl-lysine (rabbit polyclonal, Abcam, 2.5μg/ml), and anti-ubiquitin (rabbit polyclonal, Abcam, 2.5μg/ml). When immunoprecipitation was performed using anti-phosphoserine, anti-acetyl-lysine or anti-ubiquitin antibodies, membranes were probed with anti-PAX3 (mouse monoclonal, DSHB, 1/1000).