U0126

Animal experiments were conducted following standards and procedures approved by the local Animal Care and Use Committee (Tongji Medical College Experimental Animal Center, Huazhong University of Science and Technology, China). Diabetes was induced by intraperitoneal administration of streptozotocin (STZ, Sigma-Aldrich, St. Louis, MO) at 60 mg /kg, freshly dissolved in 0.05 M sterile sodium citrate (pH 4.5) on five successive days in 8-week-old male C57BL/6J mice (HFK Bioscience, Beijing, China)20 . Mice were considered diabetic if blood glucose levels determined from the tail vein using ONETOUCH glucose strips were above 300 mg/dl (16.7 mmol/L) after the last STZ injection. Mice displaying blood glucose levels above 500 mg/dl (27.7 mmol/L) received insulin Lantus (1-2U, Sanofi, Beijing, China) to avoid excessive and potentially lethal hyperglycemia. We injected a subset of diabetic mice intraperitoneally with either U0126 (LC Laboratories, Woburn, MA) (1mg/kg, dissolved in 6% DMSO-PBS) or 6% DMSO-PBS once daily starting 18 weeks after the last STZ injection until 1 day before analysis 20 . Blood and tissue samples were obtained at 26 weeks after injection of STZ.

Cells were plated in the presence of retinoic acid (5 × 10⁵/6-well) on day 1, treated with tested compounds (5 μM) plus induced Aβ-GFP expression with doxycycline (5μg/ml) on day 2, as stated. Kinase inhibitors U0126 (an inhibitor of ERK) or wortmannin (an inhibitor of PI3K) (LC Laboratories, Woburn, MA, USA) (10 μM) were added on day 6. The cells were collected on day 8 for BDNF, BCL2, BAX, total/phosphorylated TRKB, ERK, AKT, and CREB protein analysis as previously stated.

The MEK inhibitor U0126 was purchased from LC Laboratories (Woburn, MA). The JNK inhibitor SP600125 was purchased from Enzo Life Sciences, Inc./Biomol (Farmingdale, NY). Rabbit polyclonal anti-DR5 antibody was purchased from ProSci Inc. (Poway, CA). Mouse monoclonal anti-DR4 antibody (B-N28) was purchased from Diaclone (Stamford, CT). Protein-A/G plus-agarose, rabbit polyclonal anti-TRAF2 (sc-7187) and Fra-1 antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Rabbit polyclonal antibody against HA tag was purchased from Abgent (San Diego, CA). Mouse monoclonal anti-MMP1 antibody was purchased from NeoMarkers, Inc. (Union City, CA). Antibodies against actin and Flag-tag and anti-Flag M2 affinity gel were purchased from Sigma-Aldrich (St. Louis, MO). Mouse monoclonal anti-TRAF2 (#558890) and anti-FADD (#556402) antibodies were purchased from BD Biosciences (San Jose, CA). Mouse monoclonal anti-caspase-8 (#9746), rabbit monoclonal anti-caspase-8 (#4790), rabbit polyclonal ant-FADD (#2782) and all other antibodies were all purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-HA matrix agarose was purchased from Thermo Scientific/Pierce (Rockford, IL).

∆K280 tau_RD-DsRed SH-SY5Y cells in 6-well plates (5 × 10⁵/well) were treated with retinoic acid (10 µM) on day 1, followed by LMDS-1 or -2 (10 µM) and doxycycline (2 µg/mL) addition on day 2 as described. ERK inhibitor U0126 or PI3K inhibitor wortmannin (10 μM) (LC Laboratories, Woburn, MA, USA) was added on day 6. On day 8, the cells were collected for BDNF, BCL2, BAX and total/phosphorylated TRKB, ERK, AKT, CREB protein analyses as described.

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and RPMI 1640 were purchased from Thermo Scientific HyClone (UT, USA). Hybond C membrane and ECL Western blot detection system were from Pierce (Rockford, IL, USA). Rabbit polyclonal antibodies against MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, c-Fos, p-c-Fos, c-Jun, p-c-Jun, p-c-myc, c-myc, Elk1, p-Elk1, and HRP conjugated goat anti-rabbit IgG were purchased from SAB (Pearland, TX, USA). SOS1 mouse monoclonal antibody was provided from abnova Company (Taiwan). Antibodies against anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from ProteinTECH Group (Chicago, IL, USA). Rabbit polyclonal antibody against EV71/VP1 was purchased from Abcam Company (Cambridge, UK). The MEK1/2 specific inhibitor U0126 was acquired from LC Laboratories (Woburn, MA, USA) and freshly prepared as DMSO solution.