Large amount of both 20-Rh2 and 20-Ppd were coexistent in the intestine

in 4% PFA, and immunostained with polyclonal anti-TuJ1 antibody. The lengths of the longest neurites were measured by ImageJ software. Cells with neurites shorter than the diameter of its soma were excluded from the analysis. ELISA ELISA was performed using 96-well microplates coated with 1% bovine serum albumin /phosphate-buffered saline. Recombinant sAPPa, sAPPb, or C-sAPPa all at 12.2 nM final concentration in a final volume of 50 mL/wellwas plated and incubated at 4uC overnight. After washing with PBS recombinant p75NTR extracellular domain fused to human Fc chimera protein or Fc-tagged IgG chimera protein as a control was added to the plate at the indicated concentrations, and incubated for 2 h at room temperature. After incubation, the plate was washed, and goat anti-human IgG-Fc antibody was added. Horseradish peroxidase -conjugated anti-goat IgG antibody, substrate 19774075 reagent, and stop solution were used to detect protein binding. Absorbance was measured at 450 nm. Nucleofection Cortical neurons were washed and resuspended in Mouse Neuron Nucleofector Solution at a final concentration of 56106 neurons per 100 mL. The cellnucleofector solution complex and the p75NTR siRNA or control scrambled siRNA were then gently mixed and transferred into a cuvette, followed by nucleofection using the nucleofector program O-05. Immediately after electroporation, the cells were mixed with 500 mL of pre-warmed DMEM/F12 containing 10% FBS, followed by transference of the cell suspension into 3.5-cm Aglafoline supplier dishes coated with PLL. After 2 hincubation, the medium was changed to DMEM/F12 containing B27 supplement and penicillin/streptomycin. After 3 days when the expression of p75NTR was reduced by siRNA, neurons were replated on 3.5-cm dishes coated with PLL at a density of 0.56105 neurons/dish in DMEM/F12 containing 10% FBS. After another 2-h incubation, the medium was changed to DMEM/F12 containing B27 supplement, penicillin/streptomycin and 1.22 nM sAPPa 20573509 or PBS control. The neurons were incubated for 24 h, fixed in 4% PFA and immunostained with polyclonal anti-TuJ1 antibody. The lengths of the longest neurites were measured by the ImageJ software. Pull-down assay His-tagged sAPPa, sAPPb, or C-sAPPa, and Ni-agarose were incubated in binding buffer at 4uC for 1 h. Human p75NTR ECD-Fc or human IgG-Fc was added to the solution, and it was incubated at 4uC overnight. Beads were washed five times with the binding buffer. Bound complexes were eluted from beads with SDS loading buffer, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by western blotting with anti-sAPPa antibody, anti-human p75 ECD antibody and anti-human IgG-Fc antibody, or anti-sAPPb antibody. In situ binding of APP fragments to COS-7 cells COS-7 cells derived from kidney fibroblast cells of monkey were cultured and maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. The cells were plated on 3.5-cm dishes coated with poly-L-lysine at a density of 46105 cells/mL 24 h before transfection. The cells were transfected with pcDNA3 or pcDNA3-p75NTR-HA by Lipofectamine 2000 according to the manufacturer’s instructions. At 40 h after transfection, the cells were fixed in 4% Co-culture of cortical neurons with Chinese hamster ovary cells CHO cells were plated on 3.5-cm dishes coated with PLL at a density of 36105 cells/dish in DMEM/F12 containing 10% FBS 24 h before transfection. pcDNA5/FRT vector or sAPPa inserted pcDNA5/FRT vector were t