Anti malonyl lysine

Cells and tissues are lysed and homogenized with RIPA buffer (Thermo Fisher Scientific, Cat. #89900) containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Cat. # PI78447), 5 M trichostatin A (Cayman Chem, Cat. #89730), and 5 mM nicotinamide (Sigma-Aldrich, Cat. #72340). Histones were extracted from cells and tissues using a total histone extraction kit (EpiGentek, Cat. #OP-0006). Before SDS-PAGE, protein samples were heated at 70°C for 5 min. Antibodies used are listed as follows: anti-β-tubulin (Cell Signaling Technology, Cat. #2128), anti-SIRT5 (Cell Signaling Technology, Cat. #8782), anti-acetyl-lysine (Cell Signaling Technology, Cat. #9814), anti-malonyl-lysine (PTM Biolabs, Cat. #PTM-901), anti-succinyl-lysine (PTM Biolabs, Cat. # PTM-401), anti-glutaryl-lysine (PTM Biolabs, Cat. #PTM-1151), anti-ACC (Cell Signaling Technology, Cat. #3676), anti-phospho-ACC (Ser79) (Cell Signaling Technology, Cat. #3661), anti-H2B (Cell Signaling Technology, Cat. #12364), anti-KAT2A (Cell Signaling Technology, Cat. #3305), anti-HSP60 (Abcam, Cat. #ab59457), anti-Lamin A/C (Cell Signaling Technology, Cat. #2032), anti-COX IV (Abcam, Cat. #ab16056), and anti-GAPDH (Cell Signaling Technology, Cat. #5174). Ponceau S solution (Sigma-Aldrich, Cat. #P7170) was used for protein staining.

Histone malonylation levels at rDNA regions in K562 cells were examined using a Cleavage under targets and release using nuclease (CUT&RUN) assay kit (Cell Signaling Technology, Cat. # 86652) according to manufacturer’s instructions. Briefly, K562 cells treated with DMSO or 25 μM orlistat for 24 h were harvested at room temperature to minimize stress on the cells. 50,000 cells were collected for each reaction, and an additional 50,000 (same amount) cells for the input sample. Reactions for the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit (Cell Signaling Technology, Cat. mAb #9751) and the negative control Rabbit (DA1E) mAb IgG XP® Isotype Control (Cell Signaling Technology, Cat. #66362) were included in addition to anti-malonyl-lysine (PTM Biolabs, Cat. #PTM-901). Cells were mildly fixed (0.1% formaldehyde) at room temperature for 2 min, and crosslinking was stopped in 125 mM glycine. After binding of Concanavalin A beads and primary antibody, and binding of pAG-MNase enzyme, DNA was digested and diffused. DNA was then purified using DNA spin columns (Cell Signaling Technology, Cat. # 14209). 2 μl of purified DNA was used for qPCR-mediated quantification of rDNA regions. Primer pairs were designed based on a previous study,²⁶ (link) and are provided in Table S2.