Phosphor p70s6k

Cells or isolated mitochondria were lysed in the RIPA buffer containing Na₃VO₄ (1 mM) and protease inhibitors (1 μg/ml). The cell lysate proteins were separated using SDS-PAGE and transferred onto nitrocellulose membranes (Protran). After blocking with 5% non-fat milk in PBS-Tween (0.1%), the membranes were incubated with antibodies against IRS1, HIF1α (both Bethyl), BECN1, SQSTM1/p62, LC3B, phosphor-p70S6K, phospho-AMPK, AMPK, phospho-ULK1 (Ser-555), phospho-ULK1 (Ser-757), and ULK1 (all Cell Signaling), phospho-mTOR (p-mTOR, Ser-2448), mTOR, PARK2 (all ThermoFisher Scientific), and GLUT-4, cytochrome c and VDAC1 (all Santa Cruz). After incubation with the HRP-conjugated secondary IgG (Sigma), blots were developed using the ECL detection system (Pierce Biotechnology). The band intensities were visualized and quantified with ChemiDoc using the Quantity One software (BioRad Laboratories).

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).

The culture materials (medium/antibiotics/FBS) were purchased from Gibco (Grand Island, NY, USA). Chemical inhibitors for protein kinases, including LY294002 for Akt and rapamycin for p70S6K, were purchased from Sigma (St. Louis, MO, USA). Specific antibodies against SREBP-1, phosphor-Akt, Akt, phosphor-p70S6K, p70S6K, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). The control-, SREBP-1- and AMPK-specific siRNAs were purchased from Thermo (Waltham, MA, USA). All other chemicals were obtained from Sigma (St. Louis, MO, USA).

The tumor tissues were surgically excised and frozen in liquid nitrogen and then homogenized in tumor lysis buffer (Prod# 78510, Thermo, USA); after centrifugation at 12,000 g for 10 min at 4°C, the lysates were collected. The protein was quantified using a BCA Kit (Prod# 23225, Thermo, USA), separated on SDS-PAGE gels at 8%–14% polyacrylamide according to protein weight and blotted onto a PVDF nitrocellulose membrane (Bio-Rad Laboratories, USA). The membrane was blocked in 5% milk in PBST for 1 h and then probed with primary antibodies overnight at 4°C. The following primary antibodies were used: the phosphor-EGFR, EGFR, phosphor-ERK, ERK and Bcl-xL antibodies, which were purchased from Santa Cruz Biotechnology, and the mTOR, phosphor-mTOR, phosphor-4EBP1, 4EBP1, p70S6K, phosphor-p70S6K, cleaved caspase-3, caspase-3, PARP, phosphor-AKT, AKT, Jak1, phosphor-Jak1, STAT5, phosphor-STAT5, STAT3 and phosphor-STAT3 antibodies, which were obtained from Cell Signaling Technology. After washing the membranes in PBST, they were incubated with the appropriate secondary antibodies for 1 h at room temperature, washed three times in PBST and then visualized with enhanced chemiluminescence reagent, following the manufacturer's instructions (Prod# 34080, Thermo, USA).

The protein sample concentration was measured by Bicinchoninic Acid Protein Assay (ThermoFisher, Waltham, MA, USA). Proteins were subjected to SDS-PAGE separation, and the separated proteins were transferred to PVDF membrane (Pall Corporation, East Hills, NY, USA). The membrane was blocked with primary antibodies p62 (Novus Biologicals, Littleton, CO, USA), LC3 (Novus Biologicals), beclin-1 (Novus Biologicals), p70S6 kinase (p70S6K) (Cell Signaling, Danvers, MA, USA), AKT (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), mTOR (Cell Signaling), phosphor-AKT (Santa Cruz Biotechnology, Inc.), phosphor-mTOR (Cell Signaling), phosphor-p70S6K (Cell Signaling) or monoclonal antibodies against b-actin (AC-15, Sigma Aldrich). Horseradish peroxidase-conjugated goat anti-mouse IgG or anti-rabbit IgG (Jackson, West Grove, PA, USA) was used as the secondary antibody and protein-antibody complexes were visualized by enhanced chemiluminescence system (Amersham). The signals were quantified with ImageJ software (rsbweb.nih.gov/ij).

Adult male C57/BL6 mice were sacrificed at 8 weeks after TAC or sham surgery and heart samples were collected. Proteins were then extracted from these LV of hearts and assessed by western blotting analysis. The primary antibodies used were antibodies against P70/S6K, FGFR3, GAPDH (Bioworld Technology, Inc.), phosphor-P70/S6K and Caspase 3 (Cell Signaling Technology, Inc.), mTOR (Abcam, Inc), α-smooth muscle actin (α-SMA) (Abcam, Inc), atrial natriuretic peptide (ANP) (Abcam, Inc), SMARCD1 and SMARCA5 (ProteintechGroup, Inc.).