Ecl chemiluminescence system

Protein concentrations were quantified using the Bradford Assay (Bio-Rad Protein Assay 500-0006, Munchen, Germany) 5 μg of TIF extracted proteins were separated by 10% SDS polyacrylamide gel electrophoresis. PVDF membranes were blocked in Tris-buffered saline (5% non fat milk powder, 0.1% Tween20, 1 h, room temperature). Primary antibodies were diluted in the same buffer (incubation overnight, 4 °C) using: APP (cat. #2024170, 1:1000, Millipore, Billerica, MA, USA), p-APP (cat. #MABN10, 1:1000, Millipore, Billerica, MA, USA), anti Tau-5 (cat. #MABN162, 1:1000, Millipore Mab 361), p-Tau (cat. #MABN388, 1:1000, Millipore, Billerica, MA, USA), c-Jun (cat. #9165, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-c-Jun [Ser63] (cat. #9164, 1:1000, Cell Signaling Technology, Danvers, MA, USA),p-JNK (cat. #9251, 1 : 1000, Cell Signaling Technology, Danvers, MA, USA), JNK (cat. #9252, 1 : 1000, Cell Signaling Technology), anti-Actin (cat. #MAB1501, 1:5000, Millipore, Billerica, MA, USA) and at least six independent experiments were performed. Blots were developed using horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and the ECL chemiluminescence system (Promega). Western blots were quantified by densitometry using ImageQuant TL software (Amersham Biosciences, Amersham, UK) and was based on at least three independent experiments.

Proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane. Incubation with primary antibodies was overnight at 4 °C using the following: 1 : 1000 anti-MKK7 (4172, Cell Signaling Technology, Beverly, MA, USA), 1 : 1000 anti-P-MKK7 (4171, Cell Signaling Technology), 1 : 1000 anti-MKK4 (07-194 Upstate, Charlottesville, VA, USA), 1 : 1000 anti-P-MKK4 (9151, Cell Signaling Technology), 1 : 1000 anti-JNK (9252S, Cell Signaling Technology) and 1 : 1000 anti-P-JNK (9252S, Cell Signaling Technology). All P-antibodies, P-MKK7, P-MKK4 and P-JNK, are specific and recognize only the phosphorylated form of these proteins (they do not recognize cortical neuronal extracts dephosphorylated with alkaline phosphatase overnight). In a single experiment, a very large number of neurons were analyzed, providing very consistent results, and the blots were all normalized with respect to actin (1 : 10 000 anti-actin, 1501 Millipore, Billerica, MA, USA). Blots were developed using horseradish peroxidase-conjugated secondary antibodies (goat anti mouse IgG-HRP and goat anti rabbit IgG-HRP, both from Santa-Cruz Biotechnology, Santa Cruz, CA, USA) and the ECL chemiluminescence system (Promega).