Ab137133

LTA (purity of more than 98%) was provided by Sunfull Bio-tech Co., Ltd. (Changsha, China), and L-glutamine (LG, purity than 98%) was supplied by China Pharmaceutical Group Chemical Reagents Co., Ltd. (Shanghai, China).
Antibodies were used against B⁰AT1, y⁺LAT1, p-mTOR, p-S6K1, and p-S6 (ab180516, ab236669, ab137133, ab109393, and ab215214, respectively; Abcam, Cambridge, UK), β-actin, EAAT1, EAAT3, CAT-1, 4F2hc, mTOR, S6K1, and S6 (60008-1-Ig, 20785-1-AP, 12686-1-AP, 14195-1-AP, 15193-1-AP, 20657-1-AP, 14485-1-AP, and 14823-1-AP, respectively, Proteintech, IL, USA), and ASCT2 (5345, Cell Signaling Technology, BSN, MA, USA).

Primary antibody against PCAF (3305, CST), p53 (sc-126, santa cruz), p21 (sc-6246, santa cruz), p16 (sc-166760, santa cruz), Ubiquitin (sc-53509, santa cruz), phosphor-Histone H2A.X(Ser139) (#2577, CST), YAP (#14074, CST), MDM2 (BS-1223, Biogot), phospho-YAP (#13008, CST), TAZ (#83669, CST), phospho-TAZ (#59971, CST), clathrin (ab21679, abcam), caveolae (ab2910, abcam), mTOR (ab2732, abcam), p-mTOR (Ser2448) (ab109268, abcam), p-mTOR(Ser2481) (ab137133, abcam), IL-1β (sc-12742, snata cruz), IL-6 (sc-130326, santa cruz), GAPDH (#AP0063, Biogot), histone H3(BS3718, Biogot), and PA (P0500, Sigma-Aldrich) were purchased commercially.

Myocardial tissues were extracted from the left ventricle, and protein was extracted from the homogenized tissue using precooled RIPA lysis solution (Shanghai Beyotime Institute of Biotechnology, China) supplemented with 1% phenylmethylsulphonyl fluoride (Shanghai Beyotime Institute of Biotechnology, China). The BCA (Beijing Solarbio Science and Technology Co., Ltd., China) method was used to determine protein concentrations. To investigate the expression of mTOR and its phosphorylated protein (p-mTOR) in heart tissues, 50 mg of protein was separated by SDS-PAGE and transferred to PVDF membranes, which were blocked with 5% skim milk. The membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies used were anti-mTOR (1 : 2000, ab137133; ABCAm, United States), anti-phospho-mTOR (1 : 5000, ab109268; ABCAm, United States), anti-GAPDH (K106389P; Beijing Solarbio Science and Technology Co., Ltd., China), and IgG-HRP (SE134; Beijing Solarbio Science and Technology Co., Ltd., China). The membranes were then incubated with the appropriate secondary antibody for 1 h at room temperature and exposed to ECL (Beijing Solarbio Science and Technology Co., Ltd., China) in the darkroom at room temperature.

Cells were solubilized in buffer RIPA lysis buffer (Applygen). Protein concentrations were determined by using a bicinchoninic acid assay kit (Pierce Diagnostics). Proteins were separated by 10% (wt/vol) SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore) that were then blocked in 5% (wt/vol) skim milk in Tris-buffered saline with Tween 20 and incubated with the following primary antibodies overnight at 4°C: anti-human TNF-α (1:300, 60291–1-Ig, Proteintech), anti-PI3 kinase p85 (1:200, bs-0128r, Bioss), anti-phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199) (1:1000, 4228 s, CST), anti-AMPK (1:300, 10929–2-AP, Proteintech), anti-phospho-AMPKα1 (Ser473) (1:800, ab131357, Abcam), anti-mTOR (1:500, 20657–1-AP; Proteintech), anti-phospho-mTOR (S2481) (1:1000, ab137133, Abcam), anti-c-Myc (1:2000, 10828–1-AP, Proteintech), anti-HIF-1α (5 µg/ml, ab1, Abcam), anti-Glut 1 (1:1000, ab652, Abcam), anti-Glut 4 (1µg/ml, ab33780, Abcam), and anti-β-actin (1:5000, 60008–1-Ig, Proteintech). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Detection occurred using chemiluminescent visualization. Quantitative densitometric analysis of the immunoblotted bands was performed using Quantity One (Bio-Rad).

Total protein was extracted utilizing RIPA buffer (Beyotime, China), then the concentration was detected utilizing a BCA protein assay kit (Beyotime, China). The proteins were separated on 10% SDS polyacrylamide gels (Solarbio, China) and afterwards transferred to PVDF membranes (Bio-Rad, USA). Following the blocking with 5% nonfat milk, incubation of blots was done overnight at 4 °C with primary antibodies against FAP (ab207178), PI3K (ab191606), p-PI3K (ab182651), AKT (ab179463), p-AKT (ab192623), mTOR (ab134903), p-mTOR (ab137133) and GADPH (ab181602), which were purchased from Abcam (Cambridge, UK). Enhanced chemiluminescence (ECL) was used to determine the signals when the secondary antibody was incubated.