Mouse anti phospho histone h2a x γh2afx

Differentiation medium with Aβ40 peptide treatment for each condition was collected and analyzed by WB in order to determine the presence of the Aβ40 peptide in its monomeric form. For the detection of cell death, β-catenin and p-β-catenin, 50 µg of cellular extracts of differentiated cultures after Aβ peptide treatment were analyzed. Samples were boiled for 5 min, loaded on a 12% sodium dodecyl sulphate (SDS)-polyacrylamide gel, electrophoresed and transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked in PBS containing 5% nonfat dairy milk with 0.05% Tween20 (Sigma) for 1 h at RT. Blots were incubated overnight at 4 °C with primary antibodies against mouse β-actin (1:1000; Sigma), mouse anti-Aβ 4G8 (1:1000; Covance) mouse anti-phospho-Histone H2A.X (γH2AFX; 1:1000; Millipore (Burlington, MA, USA)), rabbit anti-β-catenin (1:1000; Cell Signaling) or rabbit anti-phospho-β-catenin (1:1000; Cell Signaling). The blots were developed using peroxidase-conjugated goat anti-rabbit (GARPO; 1:3000; Vector Laboratories), peroxidase-conjugated horse anti-mouse (HAMPO; 1:3000, Vector Laboratories) or goat anti-rabbit (GARPO; 1:3000; Vector Laboratories) for 1 h at RT and visualized using the ECL system (Millipore).

To determine the presence of Aβ42 peptide in its oligomeric and fibrillar forms, the differentiation medium with Aβ42 treatment for each condition was collected and analyzed by WB in each experiment. For the detection of cell death, 50 µg of protein extracts were analyzed after Aβ peptide treatment. In both cases, samples were boiled for 5 min, loaded on a 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel, electrophoresed, and transferred to nitrocellulose membranes (GE Healthcare, Little Chal-font, UK). Membranes were blocked in either PBS containing 5% nonfat powdered milk with 0.05% Tween20 (Sigma) or TBS containing 3% BSA and 0.05% Tween20 (to see the phosphorylated state) for 1 h at RT. Blots were incubated overnight at 4 °C with primary antibodies against mouse β-actin (1:1000; Sigma), mouse anti-Aβ 4G8 (1:1000; Covance, Madison, WI, USA) and mouse anti-phospho-Histone H2A.X (γH2AFX; 1:1000; Millipore, Merck). The blots were developed using peroxidase conjugated horse anti-mouse (HAMPO; 1:3000; Vector Laboratories) for 1 h at RT and visualized using the ECL system (Millipore).