FIGURE 4:

Cdk1 inhibition does not result in the intracellular accumulation of post-Golgi vesicle cargoes. (A) CDK1 and cdk1-as1 cells were treated with 1NM-PP1 or DMSO control. Cells were then pulsed with ³⁵S-methionine for 10 min and chased for the times indicated. CPY was immunoprecipitated and analyzed by SDS–PAGE. (B) Immunoblot analysis of the extracellular and intracellular pools of the post-Golgi vesicle cargo protein Bgl2. Note that inactivation of sec6-4 caused an increase in intracellular levels of Bgl2. Nap1 was used as a loading control (not shown). (C) Immunoblot analysis of the extracellular and intracellular levels of Suc2-3xHA. Cells were grown in 2% glucose containing 1NM-PP1 or DMSO control for 1 h. Suc2-3xHA was then induced by shifting cells to 0.1% glucose in the presence of 1NM-PP1 or DMSO and harvesting cells after an additional hour. Note that inactivation of sec6-4 caused a complete block in secretion of Suc2-3xHA. Nap1 was used as a loading control. (D) Immunoblot analysis of the secreted and intracellular pools of Bgl2 and Suc2-3xHA levels after actin depolymerization for 1 h using latrunculin-A. Nap1 was used as a loading control. Note that the data presented in C and D were obtained in separate experiments.