FIGURE 8:

GSK-3β kinase directly phosphorylates the P-motif of CREB-H. GSK-3β directly phosphorylates the precursor form of CREB-H in vitro. COS cells transfected with a plasmid expressing wt CREB-H under the control of the TK promoter were harvested and lysed under denaturing conditions as described in Materials and Methods. Samples were diluted into the appropriate buffer and incubated without or with phosphatase (PPase) as indicated. CREB-H was then subsequently immunopurified from these extracts, further incubated without (lanes 1 and 2) or with (lanes 3 and 4) purified GSK-3β kinase, and finally analyzed by Western blot with anti-SV5 epitope and phosphospecific anti-pS73 primary antibodies.