Anti p ir

CAA was dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, U.S.A.) to a concentration of 200 μM and stored at −20°C as a stock solution. The stock solution was diluted with Dulbecco’s modified Eagle’s medium (DMEM, HyClone, U.S.A) to a final concentration of 50 μM before use. Dexamethasone (Cell Signaling Technology, #9668) was diluted with DMEM to a final concentration of 1 μM. DMSO was added in the vehicle control group. TGF-β (240-B; R&D Systems, MN, U.S.A.) was diluted to a concentration of 6.4 ng/ml, and IL-6 (206-IL/CF, R&D Systems, MN, U.S.A.) was diluted to a concentration of 8 ng/ml before use. Insulin (Cell Signaling Technology, #9668), human IL-6 high sensitivity enzyme-linked immunosorbent assay (ELISA) Kit (Abcam, ab46042, U.S.A), human IL-17 High Sensitivity ELISA Kit (Abcam, ab46042, U.S.A), propidium iodide (Cell Signaling Technology, #9668), 4′,6-diamidino-2-phenylindole (DAPI; Cell Signaling Technology, #9668), primary antibodies including anti-IL-17RC, anti-IL-6, anti-p-IR (Cell Signaling Technology, #9542), anti-GLUT1 (Cell Signaling Technology, #9542), and monoclonal mouse anti-GAPDH (Santa Cruz, CA, U.S.A.), and horseradish peroxidase (HRP)–conjugated polyclonal goat anti-mouse and anti-rabbit (both from Santa Cruz, CA, U.S.A.) were used in the present study. Human IL-17RC adenovirus was purchased from Vector Biolabs (Malvern, PA, U.S.A.).

All chemicals used were of analytical grade and were purchased from Sigma (St. Louis, MO) unless otherwise stated. The following antibodies were used: anti-Atg7, anti-LC3, anti-p-PERK, anti-PERK, anti-p-IR, and anti-IR (Cell Signaling Technology Inc., Danvers, MA); anti-G6Pase, anti-Pck1, anti-p62, anti-p-Akt, anti-Akt, anti-p-eIF2α, anti-eIF2α, anti-GAPDH, and peroxidase goat anti-rabbit IgG and peroxidase goat anti-mouse IgG from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., CA).

The hippocampus was homogenized in lysis buffer (Sigma, St. Louis, MO, USA)]. After that, the protein was electrophoretically resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The blots were blocked with skimmed milk and incubated in anti-p-IR (1:1000; Cell Signaling Technology, USA) and anti-p-IRS-1 (1:1000; Cell Signaling Technology, USA) for 4°C overnight respectively. And then secondary HRP antibody was added. Finally, the signals were visualized by use of Enhanced Chemioluminescence kit (ECL, Amersham).