Figure 7.
Subcellular localization of GSK-3β throughout the cell cycle. (A) NIH-3T3 cells engineered to overexpress Flag-tagged wild-type cyclin D1 were arrested by serum deprivation and contact inhibition and then dispersed and restimulated with serum to synchronously enter the cell cycle. Cells plated on glass coverslips in complete medium were fixed for immunofluorecence at the indicated times after serum addition, and were stained by use of a monoclonal antibody directed to GSK-3β (top). Cellular DNA was visualized with Hoechst dye (bottom). (B) At 6 hr (G1 phase) and 16 hr (S phase) after cell cycle entry, cells from parallel cultures were disrupted, and equal quantitites of protein from nuclear and cytoplasmic fractions were separated on denaturing gels, transferred to membrane, and blotted with antibody to GSK-3β. Sites of antibody binding were detected by enhanced chemiluminescence.