The TBS-soluble fraction was aliquoted, frozen selleck chem Regorafenib in liquid nitrogen and stored at ?80��C. The pellets were resuspended in 15 volumes of TBSX and kept on ice for 30 min, followed by a second centrifugation at 100,000��g for 1 h at 4��C. The TBSX soluble fraction was aliquoted, frozen and stored as for the TBS fraction. The TBSX-insoluble pellet was resuspended in 2 mL of 70% FA and centrifuged at 100,000��g at 4��C for 1 h. The FA extracts were neutralized by the addition of 20 volumes of 1 M Tris pH 11, aliquoted and stored at ?80��C. Total protein content in TBS- and TBSX-extracts was determined via BCA assay. Total protein in FA-extracts was determined by Bradford Protein Microassay (Bio-Rad, CA, USA). A�� Measurements The levels of intracellular A��1�C40 and A��1�C42 were quantified by using a sandwich ELISA as previously described by Durairajan et al.

[15],[24], with minor modification. Equal amounts of cell or brain lysates were used for A�� quantification by sandwich ELISA. The monoclonal antibody 6E10 was used as the capture antibody by adding to ELISA plates (0.2 ��g diluted in 0.1 M Na2CO3 (pH 9.6) per well) and incubated overnight at 4��C. The plates were washed with PBS (0.05% Tween 20) and blocked with 4% BlockAce (Serotec, Raleigh, NC, USA) for 2 h at room temperature. Equilibrated protein lysates from each treatment (a 100 ��L volume was adjusted in all treatment groups by using PBS) were applied in duplicate and incubated at room temperature for 2 h with constant rotation at 30 rpm.

Biotinylated monoclonal anti-A��40 5C3 (50 ng per well) and biotinylated monoclonal 8G7 (50 ng per well) antibodies were used for detection of A��1�C40 and A��1�C42, respectively, and were diluted in 1% BlockAce and incubated for 2 h at room temperature. Plates were thoroughly washed with PBS (0.05% Tween 20), and streptavidin conjugated HRP was added for 1 h at room temperature. Finally the plates were washed four times with PBST before adding the substrate TMB for 30 min. Absorbance at 450 nm was measured in duplicate wells after addition of 2 M H2SO4. All ELISA experimental data were from three different days. Synthetic A��40 and A��42 peptides were used for construction of calibration curves, and A�� was measured in lysates. SDS-PAGE and Western Blot Analysis For Western blot analysis from cell culture or mouse brain, 10�C30 ��g total protein was separated on 10% and 15% SDS�CPAGE gels and blotted onto PVDF membranes for the detection of full length APP (Fl-APP), CTFs, sAPP��, sAPP��-sw, ��-actin and phosphorylated APP (pAPPThr668). After blocking with 5% skim milk, the blots were incubated with primary antibodies overnight at 4��C with shaking. Blots were washed AV-951 and incubated with HRP-conjugated secondary antibodies.