Ezview red protein g affinity gel beads

Co-immunoprecipitation assays of γ-secretase complex with BACE1 were modified according to a previous report [54 (link)]. Thirty-six hours after transient transfection, HEK293T cells were treated with designated chemicals or peptides for 16 h before collection. Cells were washed twice with cold PBS and lysed with IP buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10% Glycerol and 1% CHAPSO), in the absence or presence of chemicals or peptides. After centrifugation, the supernatants were incubated with anti-Flag M2 resins at 4 °C for 4 h. The resins were washed three times and eluted with SDS loading buffer before western blotting analysis. Half brains of vehicle- or XYT472B-treated APP/PS1 mice were homogenized by a glass dounce tissue grinder in buffer A (25 μM Tris-HCl, 5 mM EDTA, 5 mM EGTA, adjusted to pH 7.4) and centrifuged to remove debris and nuclei. After centrifugation at 25 000 g for 1 h, 600 mg membrane proteins were resuspended in IP buffer and incubated with antibodies at 4 °C for 16 h. For each mouse sample, equal amount of membrane fractions were incubated with 2 μg goat anti-rat IgG or MAB-1563. The antibody-antigen complexes were then incubated with pre-equilibrated Ezview Red Protein G Affinity Gel beads (Sigma) for 1 h at 4 °C. After washed, the resins were eluted with SDS loading buffer for western blotting analysis.