Sirt5

HEK293T, HCT116 and DLD1 cells were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). Inhibitors: GW5074 (Selleck Chemicals, Houston, TX, USA), leupeptin (Sigma‐Aldrich, Burlington, MA, USA) and oxamate (Sigma‐Aldrich). Antibodies: SIRT5 (catalogue #15122‐1‐AP, Proteintech, Rosemont, IL, USA), Flag M2 (catalogue #F1804, Sigma‐Aldrich), HA (catalogue #H9658, Sigma‐Aldrich), Ki67 (catalogue #ab16667, Abcam, Cambridge, MA, USA), and cleaved caspase‐3 (catalogue #9661, Cell Signaling Technology, Boston, MA, USA), optineurin (catalogue #ab23666, Abcam), β‐tubulin (catalogue #66240‐1‐Ig, Proteintech), anti‐pan‐acetyl‐lysine (catalogue #9681, Cell Signaling Technology), anti‐pan‐succinylation (catalogue #PTM‐401, PTM Biolabs, Hangzhou, China), anti‐pan‐glutarylation (catalogue #PTM‐1151, PTM Biolabs), anti‐pan‐malonylation (catalogue #PTM‐901, PTM Biolabs), LDHB (catalogue #ab85319, Abcam) and LC3 (catalogue #CY5992, Abways, Shanghai, China). Antibody specifically recognizing acetylation at lysine‐329 of LDHB was prepared commercially at Shanghai HuiOu Biotechnology Co. Ltd (Shanghai, China). Synthesized peptide TLWDIQK(Ac)DLKDL was coupled to KLH as antigen to immunize rabbit. Anti‐serum was collected after five doses of immunization.

Total protein was isolated using RIPA lysis buffer (Servicebio, China). The protein with the corresponding volume of loading buffer was boiled in a water bath for 10 min, after which the isolated proteins were separated through SDS-PAGE, transferred to a PVDF membrane, and incubated with primary and secondary antibodies including SIRT1 (Abclonal, A17307), SIRT5 (Proteintech, 15122-1-AP), and actin (Abclonal, AC038).