Bovine brain cam

Human Aβ(1–42)-HiLyte^TM-Fluor555 was purchased from AnaSpec (Freemont, CA, USA). Aβ(1–42), Aβ(25–35) and Aβ ‘scrambled’ were supplied by StabVida (Caparica, Portugal) and GenicBio Limited (Shanghai, China). His₆ was purchased from Quimigen (Madrid, Spain). Bovine brain CaM and Thrombin Clean Cleavage^TM kit were purchased from Merck-Sigma-Aldrich (Madrid, Spain).
All the other chemicals used in this work were of analytical grade and supplied by Merck-Sigma-Aldrich (Madrid, Spain) and ThermoFisher Scientific (Madrid, Spain).

All of the in vitro kinase assays used 0.4 μg of purified protein in a 10 μl total volume. The buffer contained 50 mM HEPES pH 7.5, 10 mM magnesium acetate, 1 mM DDT, 100 μM ATP, and 0.5 μCi/μl [γ-³²P] ATP in the presence of 5 mM EGTA or 0.5 mM CaCl₂ or 0.5 mM CaCl₂ with 1 μM of bovine brain CaM (Sigma). In order to determine substrate phosphorylation, two micrograms of a bovine myelin basic protein (MBP) was used as substrate. Reactions were stopped by adding SDS–PAGE sample buffer and then boiled for 1 min in water bath. Subsequently, sample reactions were analyzed in SDS–PAGE and gel as dried. The difference of substrate phosphorylation patterns at different conditions was observed by exposing gel to the Kodak autoradiography film.

The autophosphorylation assay was performed in 10 μl reaction mixture using 0.4 μg of CCaMK protein and its mutated versions. The reaction buffer contained 50 mM HEPES pH 7.5, 10 mM magnesium acetate, 1 mM DTT, 10 μM ATP and 0.5 μCi/μl [γ-³²P] ATP, in the presence of 5 mM EGTA with or without 1 μM of bovine brain CaM (Sigma); and 0.5 mM of CaCl₂ with or without bovine CaM. Samples were incubated at 30°C for 30 min. To stop the reaction, SDS–PAGE sample buffer was added, followed by boiling the samples for 2 min. Samples were separated by a 12.5% SDS–PAGE. Protein gel was then dried and exposed to autoradiography film (Kodak).