Figure 1.
Mitotic inactivation of Ikaros. (A) DNA content of exponentially growing VL3-3M2 cells (left) and mimosine- and vinblastine-treated cells (middle and right) was determined by flow cytometry. (B) The subnuclear localization of Ikaros was analyzed in asynchronous (AS) and vinblastine-arrested (G2/M) VL3-3M2 cells by confocal microscopy. DNA was visualized using propidium iodide. (C) Ikaros concentrations in asynchronous and vinblastine-arrested samples were compared by Western blot (lanes 1,2). DNA-binding activities were compared by gel shift in the absence (lanes 3,5) and presence (lanes 4,6) of calf-intestine alkaline phosphatase (20 U). (D) VL3-3M2 cells were grown in the presence of 32P-labeled orthophosphate. Asynchronous and vinblastine-arrested samples were analyzed by immunoprecipitation using Ikaros antibodies. (E) Phosphopeptide maps were generated for Ikaros from asynchronous and vinblastine-arrested VL3-3M2 cells. The five phosphopeptides that were never detected in asynchronous cells are numbered on the G2/M map.