In vitro kinase assay

To assess GSK3β Thr-7 phosphorylation by ICK and GSK3β Ser-9 phosphorylation by AKT, HA-GSK3β kinase-dead (KD) proteins were expressed in HEK293T cells, affinity-purified from cell extracts by anti-HA agarose beads. HA-GSK3β substrates (0.5–1 μg) were then incubated with active His-ICK^1-291 [28, 32] or His-Akt1/PKBα (Millipore, Dundee, UK) proteins (50–100 ng) and 100 μM [γ-³²P]-ATP (PerkinElmer, Waltham, MA, USA) in kinase buffer [50 mM HEPES (pH 7.5), 10 mM MgCl₂, 2 mM DTT, and complete protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland)] at 30°C for 20 min. After the reaction was terminated by addition of 2X SDS sample buffer, the reaction samples were processed and analyzed for radioactivity by autoradiograph as described in [28, 32]. For non-radioactive assay, the above reaction mixtures were incubated with unlabeled ATP and the reactivity of GSK3β against phospho-T7 and phospho-S9 antibodies was analyzed by Western blot.

To measure GSK3β activity against primed substrates, HA-GSK3β (WT, T7A, T8A, and S9A) proteins were expressed in HEK293T cells. After insulin (100 nM) stimulation for 60 min, recombinant GSK3β proteins were affinity-purified and then incubated with 60 μM Phospho-Glycogen Synthase Peptide-2 (p-GSP2) (Millipore, Burlington, MA, USA) [4] and 100 μM [γ-³²P]-ATP (PerkinElmer, Waltham, MA, USA) in kinase buffer at 30°C for 20 min. The reaction mixtures were spotted onto phosphocellulose paper (p81, Whatman), washed in 0.75% phosphoric acid, rinsed in acetone, and counted for radioactivity in a liquid scintillation counter.