Optineurin

Quadriceps muscle samples (n = 10) from rapamycin- and chloroquine-treated 18–20 month old wild-type and VCP^R155H/+ knock-in mice were harvested and extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Rockford, IL). Protein concentrations were determined using the Nanodrop according to the manufacturer’s protocols. Equal amount of proteins were separated on Bis-Tris 4–12% NuPAGE gels according to manufacturer’s protocols. The expression levels of proteins were analyzed by Western blotting using LC3-I/II (Novus Biologicals, Littleton, CO), p62/SQSTM1, mTOR substrates, ubiquitin, optineurin, VCP, and TDP-43 primary antibodies (Abcam, Cambridge, MA). Equal protein loading was confirmed by β actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) staining.

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.