Mtor p mtor

All inhibitors were purchased from Selleck Chemicals (Houston, TX), prepared as 10 mM stock solutions in DMSO, and further diluted with culture medium before use. Annexin V-FITC/PI apoptosis detection kit and cell cycle detection kit were purchased from KeyGen BioTECH (Nanjing, China). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DMSO, and Hoechst 33342 were obtained from Solarbio (Beijing, China). Antibodies for Src/p-Src, Mek/p-Mek, Erk/p-Erk, PI3K/p-PI3K, PDK1/p-PDK1, AKT/p-AKT, mTOR/p-mTOR, PTEN/p-PTEN(S380/T382/383), JNK/p-JNK, c-Jun, p38/p-p38, JAK2/P-JAK2, Stat3/p-Stat3, Rb/p-Rb, CDK4, CDK6, Cyclin D1, CDK2, Cyclin E1, RAD51, Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-7 were all purchased from Cell Signaling Technology (Danvers, MA, USA). Tubulin and GAPDH primary antibodies were purchased from ZSGB-Biotechnology (Beijing, China). Horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit and mouse IgG were obtained from BBI life sciences (Shanghai, China).

The whole hippocampus (ventral and dorsal) was lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors. Protein concentration was determined colorimetrically by NANODROP 2000 (Thermo Scientific). Protein lysates were separated by 10% SDS-PAGE electrophoresis and were transferred onto polyvinylidenedifluoride (PVDF) membranes. After blocking with 5% BSA for 1 hr, and the following antibodies were used: NR1(Santa Cruz Biotechnology, 5704S, 1 : 500), AKT/p-AKT (Cell Signaling Technology, 9272S, 4058s, 1:500), mTOR/p-mTOR (Cell Signaling Technology, 2972S, 2971S,1:500), p70S6 kinase p70s6k) / p--p70S6K (Cell Signaling Technology, 2708S, 9234S, 1:500), p-4EBP1 (Cell Signaling Technology, 9644S.1:1000), GluR1 (Cell Signaling Technology, 12551S, 1:1000) and β-tubulin (Epitomics, 1879-1, 1:2000). After the blots were incubated with antibodies overnight at 4 °C, they were incubated with horseradish peroxidase- conjugated secondary antibodies for 1 hr. The blots were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.). The resulting data was analyzed statistically using SPSS Statistics 20. All experiments were performed in troplicate. The final data are expressed as a ratio of the relative optical density (ROD) of the protein of interest to the ROD of β-tubulin. A p-value < 0.05 was considered significant.

HEPA 1–6 cell line and HepG2 cells were cultured as recommended by the American Type Culture Collection (Manassas, MA, USA). Rapamycin and Insulin were purchased from Sigma-Aldrich (St.Louis, MO, USA). Pertussis toxin was purchased from Calbiochem (San Diego, CA, USA). Antibodies for LC3, p-PI3K p85 (Tyr458)/PI3K, p-Akt/Akt, and p-mTOR/mTOR were from Cell Signaling (Danvers, MA, USA). Antibodies for Gαi3 and β-actin were from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies for Tnfaip8 were both produced using protein-specific synthetic peptides from AbFrontier (Seoul, South Korea) as described before^{7 (link)} and purchased from Proteintech (Rosemont, IL, USA).

Total protein lysates were prepared from pooled untreated or treated keratinocytes using RIPA buffer containing Halt protease/phosphatase inhibitors in accordance with the manufacturer’s protocol (Thermo Fisher Scientific, Rockford, IL, USA). 25 μg of protein was electrophoresed using 4–12% gradient SDS-polyacrylamide gels, transferred to PVDF membrane and blocked with 5% BSA. Primary antibodies for pEGFR/EGFR, MET, pHER2/HER2, pHER3/HER3, pAKT/AKT, STAT3, pmTOR/mTOR, B-Actin were purchased from Cell Signaling and used at 1:1000. p-MET was purchased from Abcam and used at 1:1000. Anti-rabbit HRP secondary antibodies (Cell Signaling Technology), followed by West Dura Chemiluminescence substrate (Thermo, Rockland, IL) were used for signal detection. Bands were quantified using NIH Image J and normalized to the densitometry for the respective housekeeping gene. Western blots used pooled keratinocyte protein (n = 4–6 mice/pool) and were repeated a minimum of three times.

Western blotting was performed using a standard protocol. Cells or brain tissues were sonicated and lysed with RIPA lysis buffer, and insoluble pellets were removed by centrifugation at 15,000 × g for 15 min at 4°C. Protein concentration was measured by Bradford assay (#PQ0041, MultiSciences Biotech Co., Ltd.), and the extracts were stored at -80°C until analysis. Equal amount of protein (20-40 μg) was loaded for blotting with anti-p-Akt/Akt (#4060/#4691, Cell Signaling Technology), p-mTOR/mTOR (#5536/#2983, Cell Signaling Technology), p-p70S6K/p70S6K (#9204/#9202, Cell Signaling Technology), p-4E-BP1/4E-BP1 (#2855/#9644, Cell Signaling Technology), Nrf2 (#12721, Cell Signaling Technology), NQO1 (#sc-32793, Santa Cruz Biotechnology), Cleaved Caspase-3 (#9661, Cell Signaling Technology), TH (#58844, Cell Signaling Technology), HO-1 (#sc-390991, Santa Cruz Biotechnology), Gclc (#ab190685, Abcam), Gclm (#ab126704, Abcam), and anti-β-actin (#ab8227, Abcam).