Anti atg5

The following antibodies were used for the immunoblot and immunofluorescence assays: anti-GFP (Santa Cruz Biotechnology, #sc-9996), anti-Wipi3 (Thermo Fisher Scientific, #PA5-50864), anti-Atg5 (Sigma, #A0731), anti-Lamp2 (Abcam, #ab13524), anti-RFP (MBL, #M204-3), anti-Flag (MBL, #PM020), anti-HA (Santa Cruz Biotechnology, #sc-7392), anti-Lamp1 (abcam, #ab24170), anti-VSVG (KeraFAST, #EB0010), anti-GS28 (BD, #611184), anti-GM130 (BD, #610823), anti-Tom20 (Santa Cruz Biotechnology, #sc-11415), anti-Myc (Santa Cruz Biotechnology, #sc-40), anti-Rab9 (Sigma, #R5404), anti-His (Nacalai Tesque, #04428-26), anti-LC3 (nanoTools, #0231-100), anti-p62 (MBL, #PM045), anti-Wipi2 (abcam, #ab105459), anti-actin (Millipore, #MAB1501), anti-GST (Santa Cruz Biotechnology, #sc-138), anti-Atg7 (CST, #2631), anti-GAPDH (EMD Millipore, #MAB374), anti-calbindin D-28k (Swant, #300), anti-calbindin D-28k (Sigma, #C2724), anti-GFAP (Cell Signaling Technology #12389), anti-Iba1 (Wako, #019-19741), anti-ubiquitin (CST, #3933), anti-ceruloplasmin (BD, #611488), anti-ferritin (abcam, #ab69090), anti-ferroportin (Novus, #NBP1-21502) and anti-DMT1 (Proteintech, #20507-1-AP). Enzymes used for recombinant DNA techniques were purchased from Takara Bio, TOYOBO, and New England BioLabs. All other reagents were obtained from Nacalai Tesque.

DMEM (Gibco, C11965500BT), fetal bovine serum (Gibco, 16000044), EBSS (MACGENE, CC026), FITC Annexin V apoptosis detection kit I (BD Biosciences, 556547), cell counting kit (YEASEN, QF0026), lysis buffer (Beyotime, P0013), PVDF membranes (Millipore, ISEQ00010), albumin bovine V (Solarbio, A8020), lipofectamine 2000 (Invitrogen, 11668027), Dual-Luciferase^® Reporter Assay System (Promega, E1910), and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining kit (Promega, G3250). Bafilomycin A1 was purchased from Sigma. The following antibodies were used: anti-ULK1 (Sigma-Aldrich, A7481), anti-P62 (MBL, PM045), anti-P62 (Abcam, ab56416), anti-LC3B (Sigma, L7543), anti-c-Myc monoclonal antibody (Sigma, C3956), anti-β-actin (Sino Biological, 100162-RP02-100), anti-Gapdh (Transgen, HC301), anti-ULK1 (Sigma, A7481), anti-ATG5 (Sigma, A0731), anti-ATG7 (Sigma, A2856), anti-Beclin1 (CST, 3738), anti-Tom20 (BD Biosciences, 612278), DAPI (CST, 4083), HRP Affinipure goat anti-mouse IgG (Earthox, E030110), and HRP Affinipure goat anti-rabbit IgG (Earthox, E030120). The following fluorescent secondary antibodies were used: Alexa Fluor 555-labeled donkey anti-mouse IgG antibodies (Invitrogen, A31570) and Alexa Fluor 488-labeled donkey anti-rabbit IgG antibodies (Invitrogen, A21206).

Reagent and antibody sources were as follows: acridine orange, ATRA (all-trans-retinoic acid), bafilomycin A1 (BafA1), BIX01294 trihydrochloride hydrate, DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), laminin, 3-methyladenine (3MA), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), anti-LC3 (catalog no. L7543) and anti-β-Actin-peroxidase conjugated antibody (catalog no. A3854) (Sigma-Aldrich, Munich, Germany), anti-ATG5 (catalog no. 2630), anti-ATG7 (catalog no. 2631), anti-cleaved caspase 3 (catalog no. 9661), anti-cleaved caspase 7 (catalog no. 9491), anti-cleaved PARP (poly (ADP-ribose) polymerase-1) (catalog no. 9541), anti-SOX2 (catalog no. 3579) (Cell Signaling Technology, Beverly MA, USA), anti-ULK1 (catalog no. sc-33182) (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-Beclin1 (catalog no. 612112), anti-GFAP (catalog no. 556330) (BD Pharmingen San Jose, CA, USA), anti-H3K4me3 (catalog no. 07-473), anti-H3K27me3 (catalog no. ABE44), anti-OLIG2 (catalog no. AB9610), anti-Tubulin beta III isoform (catalog no. MAB1637) (Millipore, Temecula, CA, USA), anti-H3K9me2 (catalog no. ab1220), anti-G9a (catalog no. ab40542) (Abcam, Cambridge, UK), anti-RNA Pol II (catalog no. 39097) (Active Motif, Carlsbad, CA, USA) and anti-NESTIN (catalog no. MAB1259) (R&D Systems, Minneapolis, MN, USA).

The protein lysates were isolated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto a polyvinylidene fluoride membrane (PVDF, Merck Millipore, Darmstadt, Germany), blocked with 5% buttermilk for one hour at room temperature, and cultivated with a primary antibody overnight at 4° C. Primary antibodies used included anti-LC3, anti-Atg5 (1:5000, 1:2000, and 1:2000 dilution, Sigma-Aldrich, St. Louis, Missouri, USA), anti-p62, anti-Rad51, anti-Ku70/80, anti-β actin (1:2000, 1:1000, 1:500, and 1:1000 dilutions, singly; Abcam, ab91526, ab133534, ab53126, ab8227, Cambridge, UK), γ-H2AX (1:1000 dilution, Cell Signaling Technologies, Danvers, MA, USA), and Acetylation of histone H3 (Ac-H3) (1:2000 dilution, sc56616, Santa Cruz Biotechnology, USA). The membranes were incubated with horseradish peroxidase-conjugated secondary antibody at a dilution of 1:2000 for one hour at room temperature. Protein bands were visualized using ECL Western Blotting Detection Reagents (Thermo Fisher Scientific, RJ238937, Waltham, USA) and exposed to an ECL Plus film (GE Healthcare, Piscataway, NJ, USA), and were quantified using the ImageJ software (NIH).

Consecutive 3-μM sections were cut from the TMA tissue blocks. The expression of Beclin 1, ATG5 and LC3b was analysed immunohistochemically using the following primary antibodies: anti-Beclin 1 (rabbit polyclonal; Novus Biologicals, LLC; no. NB110-87318, dilution 1:200), anti-ATG5 (rabbit polyclonal; Sigma-Aldrich Corporation; no. A0731, dilution 1:1000) and anti-LC3b (rabbit polyclonal; Abcam plc; no. ab48394, dilution 1:200). Beclin 1 and ATG5 stainings were detected by Discovery UltraMap anti-rabbit IgG in combination with ChromoMap DAB Detection Kit (Ventana Medical Systems, Inc.) and LC3b by UltraView Universal DAB Detection Kit (Ventana). After antigen retrieval (microwave oven for 10 minutes at 250 W) immunohistochemistry for Beclin 1 and ATG5 was carried out in a Discovery Ultra stainer (Ventana) and for LC3b in a Benchmark Ultra stainer (Ventana) according to the manufacturer's instructions. Normal prostatic and testicular parenchyma was chosen as positive control. For negative controls, the primary antibody was omitted. The specificity of the commercial antibodies has been thoroughly validated in former studies [31 (link)–34 (link)].

Sodium palmitate and sodium oleate were obtained from Sigma‐Aldrich. Stock solution of 50 mM of each reagent was obtained by dissolution in H₂O at 70°C. Working solutions were made by conjugation of 2 mM palmitic acid or free fatty acid (FFA) mixture (Sodium palmitate: sodium oleate = 1:2) with 2% BSA‐MEM/F‐12.³⁶ Antibodies including anti‐LC3, anti‐Beclin‐1, anti‐ATG5 and anti‐β‐actin were obtained from Sigma‐Aldrich.
Mouse hepatocyte cell line AML‐12 was obtained from Shanghai Institutes for Biological Science, CAS. Cells were maintained in MEM/F12 medium supplemented with 10% foetal bovine serum, 10 μg/ml insulin, 5.5 μg/ml transferrin, 5 ng/ml selenium and 40 ng/ml dexamethasone. Culture medium containing 0.5% lipoprotein depleted foetal bovine serum (LPDS) was used when FFA was added.

Cells were lysed in PBS that contained 1% Triton X-100 and protease inhibitor cocktail (Sigma-Aldrich). The lysates were then centrifuged at 12,000x g at 4°C for 15 min. The proteins in the lysate were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane and detected by immunoblotting. Anti-Flag and Anti-Atg5 were purchased from Sigma-Aldrich. Anti-LC3 was obtained from Nanotool (Teningen, Germany). Anti-Beclin 1 and Anti-Beclin 2 were purchased from Santa cruz Biotechnology and Novus Biologicals, respectively. Anti-p62 was purchased from Abcam.