Lc3 rabbit polyclonal antibody

Cells lysates were prepared using RIPA buffer (Beyotime, China) containing PMSF and phosphatase inhibitor (Servicebio, China). The extracts were clarified by centrifugation at 12,000 rpm for 10 min at 4°C and total protein concentrations estimated using the BCA Protein Assay Kit (Beyotime, China). Equal protein amounts were prepared in SDS-PAGE loading buffer and heated at 100°C for 5 min before protein separation by SDS PAGE and transfer to PVDF membranes (Millipore). After blocking with skim milk solution for 90 min, membranes were incubated with the indicated primary antibodies. (Src rabbit monoclonal antibody, 1:1000, CST; Phospho-Src Family (Tyr416) (D49G4) rabbit monoclonal antibody, 1; 1000, CST; t-FUNDC1 rabbit polyclonal antibody, 1:500, abcepta; p-FUNDC1 (Tyr18) rabbit polyclonal antibody, 1:500, abcepta; LC3 rabbit polyclonal antibody, 1:1000, Proteintech; P62 rabbit polyclonal antibody, 1:1000, CST; GAPDH Mouse monoclonal antibody, 1:3000, Antgene, China; Detailed antibody information is provided in Supplementary Table S1) followed by the appropriate anti-rabbit or mouse HRP-conjugated secondary antibodies. The membranes were incubated with ECL solution and protein bands visualized in an imaging system. The intensities of bands were quantified using Image J software. Uncropped Western blot images are provided in Supplementary Figure S1.

Western blotting was performed following previous laboratory procedures [15 (link), 28 , 39 (link)]. The following primary antibodies were used for blotting: anti-parkin antibody (Abcam, catalog no. ab77924; Santa Cruz Biotechnology, catalog no. sc-32282), PINK1 antibody (Novus Biologicals, catalog no. BC100–494), caspase 3 polyclonal antibody (Proteintech, catalog no. 19677-1-AP), anti-optineurin antibody [EPR20654] (Abcam, catalog no. ab213556), phospho-TBK1/NAK (Ser172) (D52C2) XP rabbit monoclonal antibody (Cell Signaling Technology, catalog no. 5483), LC3 rabbit polyclonal antibody (Proteintech, catalog no. 14600-1-AP), TOMM40 rabbit polyclonal antibody (Proteintech, catalog no. 18409-1-AP), SOD2 rabbit polyclonal antibody (Proteintech, catalog no. 24127-1-AP), COXIV rabbit polyclonal antibody (Proteintech, catalog no. 11242-1-AP), GAPDH monoclonal antibody (Proteintech, catalog no. 60004-1-Ig), beta-actin monoclonal antibody (Proteintech, catalog no. 66009-1-Ig), and alpha-tubulin polyclonal antibody (Proteintech, catalog no. 11224-1-AP). Secondary antibodies included HP-goat anti-mouse (ZsBio, catalog no. ZB-2305Beijing, China) and HP-goat anti-rabbit (ZsBio, catalog no. ZB-2301). The blots were visualized using a 5200 Chemiluminescent Imaging System (Tanon Science and Technology).