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In vitro phosphorylation of Bad by purified protein kinases. Purified recombinant His-Bad protein (1 μg) was incubated in kinase reaction buffer (10 min at 30°C) containing the following different purified, recombinant protein kinases (1 μg): AKT1/PKBα, PAK2, and the PKAC (see Materials and Methods). Phosphorylation of murine Bad (mBad) on different serine residues was determined by immunoblot analysis with site-specific anti-phospho-Bad antibodies.
