FIG. 4.

The GID binds but does not inhibit GSK-3β in vitro. (A) In vitro binding. Purified, recombinant GSK-3β (lane 1) was incubated with purified GID-2/His and bound to nickel-agarose. Eluted samples were then immunoblotted with GSK-3β antibodies. Lanes: 1, GSK-3β protein prior to column binding; 2, minimal GSK-3β binding to nickel-agarose in the absence of GID-2/His; 3, GID-2/His without GSK-3β; 4 to 6, copurification of GSK-3β with increasing amounts of GID-2/His. (B) GID-2/His does not inhibit GSK-3β in vitro. GSK-3β (25 nM) was incubated with multiple concentrations of GID protein (0.5 nM to 5.0 μM), and phosphorylation of the GS-2 peptide was assayed. Phosphorylation of tau protein was also not inhibited in vitro by up to 5 μM GID-2/His (not shown).