hTau40 was phosphorylated by CaMKII (recombinant human CaMKII alpha protein from Abcam) and GSK3β [recombinant human GSK3 beta protein (active) from Abcam]. The reaction was performed by mixing 0.2 mM hTau40 with 0.02 mg/ml kinase, 2 mM DTT, 2 mM ATP, 1 mM PMSF, and 5 mM MgCl2 in 40 mM Hepes (pH 7.4). In case of CaMKII, we additionally used 2 μM calmodulin (bovine calmodulin, recombinant from Sigma), 1 mM CaCl2, and, in case of GSK3β, 2 mM EGTA. The samples were incubated at 30°C overnight and buffer-exchanged to 50 mM NaP (pH 6.8). Protein concentrations were determined by the Bradford assay using BSA as a standard.
Bovine calmodulin
Manufactured by Merck Group
Sourced in United Kingdom, United States
Bovine calmodulin is a protein found in the cytoplasm of cells. It functions as a calcium-binding messenger protein that regulates the activity of various enzymes and cellular processes.
Automatically generated - may contain errors
4 protocols using bovine calmodulin
1
Phosphorylation of hTau40 by CaMKII and GSK3β
2
Dissolved Protein Standard Samples
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[Glu1]‐Fibrinopeptide B, bovine ubiquitin, bovine β‐lactogl‐obulin and bovine calmodulin (Sigma‐Aldrich, Gillingham, Dorset, UK) were each dissolved at a concentration of 0.2 mg/mL in 50 mM ammonium acetate buffer. Fifteen percent TFE (Sigma‐Aldrich) was added as stated.
3
DAPK Kinase Activity Assay
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Endogenous DAPK was immunoprecipitated as described above using protein A-agarose beads bound to a rabbit polyclonal antibody raised against the DAPK C-terminal tail region (sc-10805, Santa Cruz). Beads were incubated for 30 min at 30°C in 30 μl reaction buffer (50 mM HEPES pH 7.5, 8 mM MgCl2, 2 mM MnCl2 and 0.1 mg/ml BSA, 0.5 mM CaCl2, 50 mM ATP, 2 mg MLC (Sigma-Aldrich))containing 15 mCi [γ-32P]ATP, and the indicated amounts of bovine calmodulin (Sigma-Aldrich) or 5mM EGTA. Samples were analyzed by immunoblotting and autoradiography. An in vitro kinase assay as described previously [53 (link)].
4
Calmodulin-Binding Peptide Interaction Assay
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The peptide corresponding to the putative calmodulin-binding site in FvUGT1 (aa 230–249) was synthesized by Genemed Synthesis, Inc. (San Antonio, TX, USA). Mixes (total volume 30 μL) containing 240 pmol (4 μg) of bovine calmodulin (Sigma, St. Louis, MO, USA) and different amounts of purified synthetic peptide in 100 mm Tris–HCl (pH 7.2), plus either 1 mM CaCl2 or 1 mM EGTA, were incubated for 1 h at room temperature. The mixes were analyzed by nondenaturing PAGE as described [59 (link)].
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