IPs were performed as previously described (64): Cells were disrupted with a Fastprep (MP) Biomedicals, and proteins were precipitated on appropriately coated Dynabeads (Invitrogen) following the instructions of the manufacturer for 2 hours and washed four times in NP-40 buffer [6 mM Na2HPO4, 4 mM NaH2PO4, 1% Nonidet P-40, 150 mM NaCl, 2 mM EDTA, and 50 mM NaF supplemented with complete EDTA-free and PhosSTOP tablets (Roche)] and once in kinase buffer [10 mM MgCl2, 50 mM tris-HCl (pH 7.5), 2 mM dithiothreitol, 1 mM EDTA, and 1 mM EGTA] before the kinase reaction in kinase buffer supplemented with 1 μl of γ32P-ATP (adenosine triphosphate; 3000 Ci/mmol), 100 μM ATP, and 1 μg of Elp4 peptide (corresponding to 1 μl). Peptides were synthetized by Eurogentec with the following sequences: Elp4, SAGEDNHSSPPSKN; Elp4, S118-P SAGEDNHSSPPS(Ph)KN; Elp4, S114A S118-P SAGEDNHASPPS(Ph)KN; and Elp4, S115A S118-P SAGEDNHSAPPS(Ph)KN. The assay was performed for 30 min at 30°C and separated on tris-Tricine gel (Bio-Rad). GST-Gsk3 fusion proteins were expressed from pGEX4T1 and purified following the instructions of the manufacturer (GE Healthcare).

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