Proteases inhibitors cocktail p8340

Brain tau kinase assay was done according to our previous protocol (Planel, Miyasaka et al. 2004 ) with some modifications. 1µg of full length recombinant tau (reference T1001-2, R Peptide, Watkinsville, GA, USA) was prepared in sample buffer containing 5µg of RIPA cortex lysate. This reaction was incubated for 60 minutes in triplicates for each temperature (37°C, 40°C and 42°C) and terminated by adding one volume of sample buffer (NuPAGE LDS; Invitrogen, Carlsbad, CA) containing 5% of 2-β-mercapto-ethanol, 1 mM Na 3 VO 4 , 1 mM NaF, 1 mM PMSF, 10 μl/ml of Proteases Inhibitors Cocktail (P8340; Sigma-Aldrich, St. Louis, MO, USA), and heated for 10 min at 95 °C. A negative control without ATP was processed in parallel following the same procedure in duplicate for each temperature. Samples were analyzed by SDS-PAGE and western blotting as described above. Immunoblot was analyzed using anti tau PS404 antibody (see section antibodies) since this epitope is phosphorylated by most of the major tau of kinases (Planel 2002) . The signal was normalized to total tau. Immunoreactive bands were visualized using ImageQuant LAS 4000 imaging system (Fujifilm USA, Valhalla, NY, USA), and densitometric analyses were performed with Image Gauge analysis software (Fujifilm, Valhalla, NY, USA). Images levels were adjusted.