Anti atg13

MRT compounds were synthesized as described (21 (link)). AZD8055 was from Selleck Chemicals, and bafilomycin A1 was from Enzo Life Sciences. Rabbit anti-ULK1, anti-ATG5, and anti-ATG13 were from Sigma. Rabbit anti-phospho-Ser-757 ULK was from Cell Signaling Technology, and rabbit anti-phospho-Ser-318 ATG13 was from Abnova. Mouse anti α-tubulin was from Merck-Millipore. Mouse anti-LC3 for immunofluorescence was from MBL International, and sheep anti-LC3, used for immunoblotting, was generated by the Division of Signal Transduction Therapy, University of Dundee, from recombinant full-length human GST-LC3b and affinity-purified.

anti-pS757 ULK1, anti-pS555 ULK1 and anti-ULK1 were from CST, anti-pS318 ATG13 from Novus, anti-ATG13 from Sigma–Aldrich, anti-α-Tubulin from Proteintech and sheep anti-LC3b (S400D) from MRC PPU Reagents and Services. All antibodies were used for Western blotting at 1:1000 dilution apart from the LC3b antibody which was used at 1:500. HRP-conjugated secondary anti-sheep, anti-rabbit and anti-mouse antibodies were purchased from Thermo Scientific and used in 1:10 000 dilution. For immunofluorescence, anti-WIPI2 (Bio-Rad), anti-ULK1 (CST) and anti-LC3 (MBL) antibodies were used in 1:500 dilution. Secondary antibodies for immunofluorescence (anti-mouse Alexa Fluor® 488 and anti-rabbit Alexa Fluor® 594) (Thermo Scientific) were used in 1:1000 dilution.

Human aorta specimens were obtained from a recipient of cardiac transplantation with ischemic cardiomyopathy and severe coronary artery disease. Normal human aorta specimens obtained from a donor of cardiac transplantation with normal coronary artery were used for comparison. Human tissue collection was approved by our Institutional Review Board (IRB approval number: A-ER-103-043). For double immunofluorescent staining, the sections were stained for anti-human CD31 (1:50; GeneTex, Irvine, CA) and anti-LC3 (1:500; MBL) or anti-ATG13 (1:200; Sigma-Aldrich) antibodies, followed by incubation with respective fluorescent secondary antibodies (1:200; Invitrogen). Isotype IgG control antibodies were used as negative controls to confirm the specificity of respective primary antibodies (Calbiochem, San Diego, CA) in human and mouse aortic specimens (Supplement Figure II). Nuclei were counterstained with DAPI. The fluorescent signals were captured using a fluorescent microscope (BX61, Olympus).