Purified β-adducin (human sequence) was generated in Escherichia coli with a 6xHis construct (Topo PCR T7/NT construct, Invitrogen). Expression from an O/N culture (optical density (OD)=0.4) was induced by 1 mM isopropyl-β-D -thiogalactoside for 6 h, after which bacterial pellets were collected by centrifugation for 30 min at 2,000 r.p.m. Pellets from 50-ml culture were lysed in 1 ml lysis buffer (50 mM sodium dihydrogen phosphate, 300 mM NaCl, 10 mM imidazole and 1 mg ml−1 lysozyme; pH 8.0), sonicated for 2 × 10 s and incubated for 30 min on ice. Next, lysates were cleared by centrifugation for 10 min at 13,000 r.p.m. and the His-tagged protein was enriched using pre-equilibrated Ni-NTA spin columns (Qiagen) according to the manufacturer's instructions. Eluted fractions were desalted and concentrated on amicon ultra-2 centrifugal filter devices 50K (Millipore).
Purified β-adducin (final concentration: 1 μM), DARPP-32 (1 μM), CK2α2β2 (100 ng per assay; New England Biolabs) and calmodulin (1 μM Sigma) were used for in vitro assay of Ser97 phosphorylation of DARPP-32. DARPP-32 and β-adducin, negative control or calmodulin were preincubated for 5 min in CK2 buffer (final concentration 50 mM HEPES, 20 mM MgCl2, 150 mM NaCl and 100 μM ATP; pH 7.5). Then, CK2α2β2 was added and the samples were incubated for 30 min at 30 °C and subsequently immunoblotted for pSer97 and total DARPP-32.
Purified β-adducin (final concentration: 1 μM), DARPP-32 (1 μM), CK2α2β2 (100 ng per assay; New England Biolabs) and calmodulin (1 μM Sigma) were used for in vitro assay of Ser97 phosphorylation of DARPP-32. DARPP-32 and β-adducin, negative control or calmodulin were preincubated for 5 min in CK2 buffer (final concentration 50 mM HEPES, 20 mM MgCl2, 150 mM NaCl and 100 μM ATP; pH 7.5). Then, CK2α2β2 was added and the samples were incubated for 30 min at 30 °C and subsequently immunoblotted for pSer97 and total DARPP-32.