Vamp8

The antibody for Sp56 was kindly gifted from Prof. Fei Gao. Other antibodies were purchased according to the following information: Afaf (Abcam, Cambridge, UK, ab121470); Drp1 (BD bioscience, Franklin Lakes, NJ, USA, 611113); GM130 (Abcam, ab52649); LAMP1 (Sigma-Aldrich, St. Louis, MO, USA, L1418); LC3B (Sigma-Aldrich, L7543); Nrdp1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-365622); OPTN (Proteintech, Rosemont, IL, USA, 10837-1-A); Parkin (Sigma-Aldrich, P6248); p62 (Cell Signaling Technology, Danvers, MA, USA, 88588); SIP/CacyBP (Santa Cruz, sc-166455); Sox9 (Abcam, ab185966); Tim23 (BD bioscience; 611222); VAMP8 (Abcam, ab76021); Ubiquitin (Cell Signaling Technology, 3936S); and β-actin (Sigma-Aldrich, A5441). Peroxidase-conjugated anti-mouse IgG (ZSGB-BIO, Beijing, China, ZB-5305, 1:5000), anti-rat IgG (ZSGB-BIO, ZB-2307, 1:4000), or anti-rabbit IgG (ZSGB-BIO, ZB-5301, 1:3000) was used as the secondary antibody. The protein bands were visualized using the ECL detection system (Millipore, Burlington, MA, USA).

LysoTracker Red (LTR, L12492) and MitoTracker Green (MTG, M7514) were obtained from Invitrogen (Carlsbad, CA), whereas autophagosome dye (DAPGreen, NY561) and autolysosome dye (DALGreen, PF727) were obtained from Dojindo Laboratories (Kumamoto, Japan). Carbonyl cyanide m-chlorophenylhydrazone (045200), rapamycin (12921), and oligomycin A (41105) were obtained from Thermo Fisher Scientific (Grand Island, NY). Penicillin–streptomycin (15140163, 10,000 units/mL), fetal bovine serum (FBS; 26140079), horse serum (26050-088), Dulbecco’s modified Eagle’s medium (DMEM; 11965118), F12K cell media (21127-022), phenol-free medium (1894117), and other cell culture reagents were obtained from Gibco BRL (Grand Island, NY). Primary and secondary antibodies used were GAPDH (5174, Cell Signaling Technology, Danvers, MA), FIP200 (12436, Cell Signaling Technology), ATG13 (13273, Cell Signaling Technology), VAMP8 (ab76021, Abcam, Cambridge, MA), HRP-linked anti-rabbit IgG (7074, Cell Signaling Technology), siRNAVAMP8 (VAMP8-HSS112731, 1299001, Invitrogen, Carlsbad, CA), and silencer negative control siRNA (AS020K25, Ambion, Austin, TX), Coupa-lyso probe was grifted from Zijian Guo lab (Nanjing University, Nanjing, China).