Anti p ulk1

Cells were harvested and lysed with 20 mM Tris, 135 mM NaCl, 10% glycerol, 1% NP40, pH 6.8. Protein content of cell lysates was measured using Pierce BCA Protein Assay (Thermo Scientific, Waltham, MA, USA, 23225). During each procedure equal amounts of protein were used. SDS-PAGE was done by using Hoefer miniVE (Amersham, UK). Proteins were transferred onto Millipore (Billerica, MA, USA) 0.45 µm PVDF membrane. Immunoblotting was performed using TBS Tween (0.1%), containing 5% non-fat dry milk for blocking membrane and for antibody solutions. Loading was controlled by developing membranes for GAPDH or dyed with Ponceau S in each experiment. The following antibodies were applied: antiLC3B (SantaCruz, Santa Cruz, CA, USA, sc-16755), antiPARP (Cell Signaling, Danvers, MA, USA 9542S), antiGADD 153 (SantaCruz, sc-7351), antiCREB-2 (SantaCruz, sc-200), antiP-c-Jun (Cell Signaling, 9261S), antic-Jun (Cell Signaling, 9165S), antiPERK (Cell Signaling, 3192S), antiULK1 (Cell Signaling, 8054S), antiP-ULK1 (S555) (Cell Signaling, 5869S), antieIF2α (Cell Signaling, 9722S9), antiP-eIF2α (Cell Signaling, 9721L), and antiGAPDH (Santa Cruz, 6C5), HRP conjugated secondary antibodies (SantaCruz, sc-2020 and Cell Signaling, 7074S, 7076S).

Following primary antibodies were used in the present study: anti-GAPDH (1:5000; arigo, ARG10112), anti-TH (1:1000; BD biosciences, 612300), anti-α-synuclein (1:1000; BD bioscicences, 610786), anti-p-TrkB (1:1000; abcam, ab229908), anti-trkB (1:1000; Cell Signaling Technology, 4603 T), anti-LC3A/B (1:1000; Cell Signaling Technology, 12741S), anti-P62 (1:1000; proteintech, 18420-1-AP), anti-beclin-1 (1:1000; Cell Signaling Technology, 3738S), anti-p-ERK1/2 (1:1000; Cell Signaling Technology, 4370S), anti-ERK1/2 (1:1000; Cell Signaling Technology, 4695), anti-p-LKB1 (1:1000; Cell Signaling Technology, 3482S), anti-LKB1 (1:1000; Cell Signaling Technology, 3047), anti-p-AMPK (1:1000; Cell Signaling Technology, 2535S), anti-p-mTOR (1:1000; Cell Signaling Technology, 5536T), anti-AMPK (1:1000; Cell Signaling Technology, 2532) and anti-p-ULK1 (1:1000; Cell Signaling Technology, 5869).

Antibodies used were anti-FASN (3180; Cell Signaling, Switzerland), anti-LC3B (WB: NB600-1384, Novus biological, Switzerland; IF: 3868; Cell Signaling, Switzerland) anti-LAMP1 (14-1079-80; Thermofisher, Switzerland), anti-p62 (HPA003196; Sigma-Aldrich, Switzerland), anti-TFEB (4240; Cell Signaling, Switzerland), anti-p-TFEB (Ser 211) (37681S, Cell Signaling, Switzerland), anti-p-AKT (Ser473) (4060S, Cell Signaling, Switzerland) anti-PTEN (9552, Cell Signaling, Switzerland), anti-ULK1 (4776; Cell Signaling, Switzerland), anti-p-ULK1 (Ser757, equivalent to Ser758 of human ULK1) (6888; Cell Signaling, Switzerland), anti-ATG13 (6940; Cell Signaling, Switzerland), anti-pATG13 (Ser318) (600-401-C49; Rockland, Switzerland), anti p-mTOR (Ser2448) (5536; Cell Signaling, Switzerland), p4E-BP1 (Thr37/46) (2855; Cell Signaling, Switzerland), anti-α-tubulin (3873; Cell Signaling, Switzerland), anti-cleaved PARP (9541; Cell Signaling, Switzerland), anti yH2AX (2577; Cell Signaling, Switzerland) and anti-CD11b-PE (R0841; Dako, Switzerland).

NaB and SAHA were purchased from Sigma Aldrich (Deisenhofen, Germany). The FOXO1 inhibitor AS1842856 (AS) was obtained from Selleck, and the adenosine monophosphate-activated protein kinase (AMPK) inhibitor was purchased from MCE. The monoclonal anti-ubiquitin, anti-acetylation, anti-Snail, anti-Smad2, anti-Smad3, anti-Smad2/3, anti-p-Smad2/3, anti-FOXO1, anti-LC3B, anti-P62, anti-ULK1, anti-p-ULK1, anti-LC3, anti-GAPDH, and anti-vimentin antibodies and the secondary anti-mouse or anti-rabbit antibody conjugated to horseradish peroxidase (HRP) were acquired from Cell Signaling Technology (MA, USA). The secondary anti-rabbit antibody conjugated to fluorescein isothiocyanate (FITC), Protein A/G Sepharose and anti-AMPK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-AMPK and anti-FN were acquired from Abcam. A Millicell chamber (8 mm) was purchased from Millipore (BD Biosciences, USA). The Annexin V, FITC apoptosis detection Kit S was obtained from Japan Tongren Chemical. SYBR Premix ExTaq II was a product of TaKaRa (TBI, Japan). siRNA FOXO1 and siRNA ULK1 were purchased from RIBO, and Lipofectamine 3000 was purchased from Invitrogen (Carlsbad, CA, USA). 4,6-diamidino-2-phenylindole (DAPI) dye was obtained from FUDE company.

Proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred on either polyvinylidene fluoride membranes or nitrocellulose membranes (Amersham Bioscience, Piscataway, NJ, USA). Membranes were probed using the following antibodies: anti-p62 (Abcam, Cambridge, UK); anti-Atg5, anti-P-Atg14 (Ser29), anti-Beclin-1, anti-FLIP, anti-LC3, anti-NEDD4, anti-mTOR, anti-P-mTOR (Ser2448), anti-ULK1, anti-P-ULK1 (Ser757), anti-V5, anti-Vps34 (Cell Signaling Technology); anti-BECN1, anti-GFP, anti-HA (Santa Cruz Biotechnology, Heidelberg, Germany); and anti-β-Actin, anti-Tubulin (Sigma-Aldrich). Secondary antibodies were horseradish peroxidase-conjugated anti-mouse or anti-rabbit (Bio-rad, Hercules, CA, USA). Membranes were washed with Tris-buffered saline (Medicago, Danmarks-Berga, Uppsala, Sweden) with 0.1% Tween-20 (Sigma-Aldrich) and developed through the chemiluminescence system (Amersham Bioscience) on the ChemiDock image analyser (Bio-Rad), which was also used for densitometric quantifications.

GSK2606414 (S7307) was ordered from Selleck (Pittsburgh, PA, USA), and LPS (HY-D1056) was ordered from MedChemExpress (Monmouth Junction, NJ, USA). The antibodies, including anti-LC3 (12741), anti-LC3-Ⅱ (3868), anti-P62 (5114), anti-ATG5 (12994), anti-ATG12 (4180), anti-Beclin-1 (3495), anti-p-PERK (Thr980, 3179), anti-p-eIF2α (Ser51, 3398), anti-P65 (8242), anti-p-P65 (Ser536, 3033), anti-Lamin B1 (13435), anti-LAMP1 (9091), anti-IκBα (4814), anti-p-IκBα (Ser32, 2859), anti-mTOR (2983T), anti-p-mTOR (ser2448, 5536), anti-ULK1 (6439), anti-p-ULK1 (ser555, 5869), anti-p-ULK1 (ser757, 14202), anti-AKT (2920), and anti-p-AKT (Ser473, 4060) were ordered from Cell Signaling Technology (Danvers, MA, USA); anti-TOM20 (11802-1-AP), anti-GM130 (11308-1-AP), anti-Bip (11587-1-AP), anti-ATF6 (24169-1-AP), anti-PERK (24390-1-AP), anti-eIF2α (11170-1-AP), anti-ATF4 (10835-1-AP), anti-XBP1 (25997-1-AP), anti-β-actin (20536-1-AP), and anti-GFP (50430-2-AP) were ordered from Proteintech (Rosemont, IL, USA); anti-Calnexin (C4731) was ordered from Sigma-Aldrich (St. Louis, MO, USA); and anti-TIA-1 (sc-166247) was ordered from SCBT (Dallas, TX, USA).