Figure 5

GSK3 activity does not vary between S and G2 phases: NIH3T3 cells were synchronized with thymidine treatment and released by thymidine removal for the indicated times. Cells remain in S phase for the first four hrs after thymidine removal and then enter G2 phase thereafter. (A) Cell lysates were collected at the indicated times following thymidine removal, and subjected to Western analysis for GSK3β phosphorylated on position 9, together with total GSK3 levels (upper panel). The results were quantitated, and the ratio of phosphorylate to non-phosphorylate GSK3 is presented (lower panel). (B) GSK3 protein was immunoprecipitated from cell lysates collected at the indicated times following thymidine release and the GSK3 protein assayed for kinase activity against a synthetic substrate. This figure represents the combined results of 10 different analyses, normalized to the level of GSK3 activity in thymidine-blocked cells. For comparison, S phase lysates were treated with 50 mM LiCl.