FIG. 4.

In vitro phosphorylation of GSK-3 by purified PKA. The phosphorylation reaction mixture consisted of 4 U of purified PKA catalytic subunit, 1 μg of GSK-3β fusion protein, and 100 μM ATP. Reactions were carried out in the absence and presence of the PKA inhibitor H-89 (5 μM) and the PKI inhibitor (2.5 μM). The phosphorylation reaction was allowed to proceed for 30 min at 30°C and was stopped by adding 3× SDS sample buffer. The phosphorylation of GSK-3β (top) was measured by Western blotting with phospho-GSK-3β (Ser9) antibody. The membrane was stripped and reprobed with a monoclonal phosphorylation-independent antibody to GSK-3β (bottom).