Phosphor mtor

Cells were plated in 6-well tissue culture plates at 80% confluence and incubated overnight. Cell lysates were obtained from transduced cells using cold radioimmunoprecipitation assay buffer [20 mmol/l Tris-HCl (pH 8.0), 100 mmol/l NaCl, 10% glycerol, 1% NP40, 0.5% sodium deoxycholate]. Twenty micrograms of protein mixture were separated on 10% SDS-PAGE gels and wet transferred to nitrocellulose membrane (GE Healthcare Life Sciences) and then blocked for 1 h at room temperature in TBS-T buffer [50 mmol/l Tris-HCl (pH 7.5), 150 mmol/l NaCl, 0.1% Tween-20] containing 5% non-fat milk. Membranes were then incubated overnight at 4°C or 1 h at room temperature with the respective primary antibodies: anti-CIP2A (1:500), and anti-actin (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphor-mTOR (1:1,000), mTOR (1:1,000), phospho-AKT (S473) (1:1,000), (Cell Signaling Technology, Danvers, MA, USA). Anti-mouse or anti-rabbit secondary antibody-conjugated to horseradish peroxidase was used to visualize the stained bands with an enhanced chemiluminescence visualization kit (both from Santa Cruz Biotechnology).

The protein content in each sample was determined by bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL). Quantified each sample concentration to 60–80 μg and add 4 × SDS sample dye and then denatured sample for 10 min at 95°C. Proteins were fractionated on SDS-PAGE, transferred onto Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham, Little Chalfont, UK) and detected with antibodies against IDO (Thermo Scientific, Rockford, IL), the mammalian target of rapamycin (mTOR) (Cell Signaling, Danvers, MA), phosphor-mTOR (Cell Signaling), protein kinase B (AKT) (Santa Cruz Biotechnology, Inc. Santa Cruz, CA), phosphor-AKT (Santa Cruz Biotechnology, Inc.), p70S6K (Cell Signaling), phosphor-p70S6K (Cell Signaling), microtubule associated protein 1 light chain 3 (LC3) (Novus Biologicals, Littleton, CO), Beclin (Novus) and β-actin (Sigma Aldrich). Rabbit anti-mouse IgG-peroxidase antibody (Sigma Aldrich) and goat anti-rabbit IgG-peroxidase antibody (Sigma Aldrich) were used as the secondary antibody and protein-antibody complexes were visualized by enhanced chemiluminescence system (Amersham) [56 (link)]. The signals were quantified with ImageJ software (rsbweb.nih.gov/ij) [57 (link)].

Rotenone and TA of the highest purity were bought from Sigma Aldrich, Missouri, USA. The RIPA buffer and antibodies for glial fibrillary acidic protein (GFAP), COX-2, and iNOS were purchased from Sigma Aldrich, (St. Louis, MO, USA). The protease and phosphatase inhibitor combination were obtained from Thermo Fisher Scientific (Waltham, MA, USA). A polyclonal rabbit anti-tyrosine hydrolase antibody was obtained from Merck, Darmstadt, Germany. The antibodies, LC3, p62, mTOR, phosphor mTOR, and p70s6, were purchased from Cell Signaling Technology, (Danvers, MA, USA). The apoptotic polyclonal markers, Bax and Bcl-2, were purchased from (Cambridge, Abcam, MA, USA), and the monoclonal mouse anti-synuclein antibody was obtained from BD Biosciences, (San Jose, CA, USA). Anti-Iba-1 antibody was obtained from Wako Chemicals, Richmond, VA, USA. The fluorescent secondary antibody, Alexa Flour 488, was purchased from Thermo Fischer Scientific, (Waltham, MA, USA). The biotinylated secondary goat anti-rabbit antibody was obtained from Jackson immune research laboratory, (Baltimore Pike, West Grove, PA, USA). The biochemical assays were performed using commercially available kits. The additional compounds utilized in these tests were all purchased from regional vendors and were of analytical-grade quality.

Rotenone, Valeric acid, RIPA lysis buffer, antibodies against inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2) and glial fibrillary acidic protein (GFAP) were procured from Sigma-Aldrich, St. Louis, MO, USA. Protease and phosphatase inhibitor cocktail were procured from Thermo Scientific, USA. Anti-tyrosine hydrolase (Polyclonal rabbit) antibody was obtained from Merck, Germany. The following antibodies were purchased from Cell Signalling Technology, Beverly, MA, USA: LC3, p62, mTOR, phosphor mTOR and p70S6K. Apoptotic polyclonal markers (Bax and Bcl-2) were obtained from Abcam, USA. Monoclonal mouse anti-α-synuclein antibody was purchased from BD Biosciences, San Jose, CA, USA. Anti- Iba-1 antibody was purchased from Wako chemicals, Richmond, VA, USA. Fluorescent secondary antibodies (Alexa Flour 488) were purchased from Thermo Fischer Scientific, Waltham, MA, USA. Biotinylated goat anti-rabbit secondary antibody was purchased from Jackson Immunoresearch, West grove, PA, USA. Biochemical assays were performed using commercially available kits. All other chemicals used in this experiments were provided by local commercial sources (analytical grade quality).

Muscle tissues and myotubes were homogenized in lysis buffer containing 20 mM HEPES (pH 7.2), 150 mM NaCl, 0.5% Triton X-100, 0.1 mM Na₃VO₄, 1 mM NaF, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), and 5 mg/ml aprotinin (Sigma-Aldrich). The lysates were centrifuged at 15,000×g for 20 min at 4 °C, and the supernatants were subjected to SDS-PAGE followed by immunoblot analysis. Antibodies used are as follows: 4EBP1 (#9452), ATG5 (#12994), ATG7 (#8558), ATG12 (#4180), ATG16L (#8089), beclin1 (#3495), eIF2α (#9722), IRE1α (#3294), mTOR (#2983), p65 (#6956), PERK (#3192), S6K (#9202), SEK (#9152), SQSTM1 (#8025) phospho-4EBP1 (#9459), phospho-AKT S473 (#9271), phospho-GSK-3β (#9327), phosphor-mTOR (#5536), phospho-eIF2α (#9721), and phospho-PERK (#3179), phospho-JNK (#9251), phospho-SEK (#9151), phospho-S6K (#9206) from Cell Signaling Technologies; phospho-IRE1α (ab48187) from Abcam; and HA (sc-805), AKT (sc-1618), ATF6 (sc-22799), MYH (B-5, sc-376157), GSK-3β (sc-7291), and FABP3 (sc-58274) from Santa Cruz Biotechnology; and JNK (51-1570) from BD Bioscience; Laminin (L9393) from Sigma-Aldrich. The anti-GAPDH antibody was developed in our laboratory. Data were collected using Automatic X-ray Film Processor (JP-33, JPI America) or iBright FL1500 (Invitrogen).

To analyze the protein expression levels, cell lysis was performed in buffer containing 0.5% Nonidet P40 (Roche, Mannheim, Germany, 11754599001), 100 mM Tris-HCl (pH 7.4), 300 mM NaCl, protease, and phosphatase inhibitors [20 (link),21 (link)]. Proteins were resolved by SDS-gel electrophoresis, and the blots were probed with the following antibodies: HRP-β-actin (AC-15) (ab49900, 1:20,000), cathepsin B (CTSB, ab58802, 1:1000), and LAMP1 (#24170, 1:1000) from Abcam, Cambridge, UK; CTSB (H-5) (sc-365558, 1:1000), CTSC (D-6) (sc-74590, 1:1000), CTSD (D-7) (sc-377299, 1:1000), LAMP1 (H4A3) (sc-20011, 1:1000) and LAMP2 (H4B4) (sc-18822, 1:1000) from Santa Cruz Biotechnology, Dallas, DX, USA; EEA1 (#2411, 1:1000), EGFR (D38B1) (#4267T, 1:1000), Rab5A (#2143, 1:1000), Rab7 (D95F2) (#9367, 1:1000), phosphor-AKT (ser473, #9271), phosphor-ERK1/2(Thr202/Tyr204, #9101), phosphor-mTOR (ser2448, #2971, 1:1000), phosphor-S6 ribosomal protein (D57.2.2E) (ser235/236, #4858, 1:2000), α-tubulin (DM1A) (TUBB, #3873S, 1:1000) from Cell Signaling Technology, Danvers, MA, USA; GAPDH (MAB374, 1:5000) from Merk-Millipore, Burlington, MA, USA; CTSL (CPL33/1) (C4618, 1:1000) and LC3 (L7543, 1:5000) from Sigma-Aldrich; and SQSTM1/p62 (#H00008878, 1:5000) from Abnova, Taibei, China.

Antibodies against phosphor-AMPKα, AMPKα, phosphor-ACC, ACC, phosphor-mTOR, mTOR, phosphor-p70S6K, p70S6K, phosphor-4E-BP1, 4E-BP1, phorphor-NFκB p65, NFκB p65, IκBα, COX2, phosphor-STAT3, STAT3, Mcl-1, Bcl-xL, Bim, Bak, Bax, Bid, Puma and Bad were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Bcl-2 and Protein A/G Agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Metformin and aspirin as well as interleukin-6 (IL-6) were purchased from Sigma-Aldrich (St Louis, MO, USA). AZD-8055 was purchase from Invitrogen (Carlsbad, CA, USA).