Fig 4.

PHF8 protein levels can be modulated by changing CDC20 levels. (A) The empty vector control or increasing amounts of HA-CDC20 (0.2, 0.5, and 1.0 μg) were transfected into HEK293T cells, and lysates were immunoblotted for endogenous PHF8. (B) A total of 20 nM the control or two different siRNAs targeting different regions of CDC20 was transiently transfected into HeLa cells twice at 0 h and 24 h. Seventy-two hours after the first transfection, cells were harvested, lysed, and subjected to immunoblotting. (C) HEK293T cells were transfected with 0.5 μg of the empty vector control or the HA-CDC20 plasmid, treated with 100 μg/ml of cycloheximide, and harvested at 0, 2, 4, or 8 h. Lysates were immunoblotted for PHF8 and cyclin B1, and bands were quantitated by ImageJ and normalized against actin and the 0-h sample (I). Means and standard deviations of normalized PHF8 (II) and cyclin B1 (III) levels from triplicate experiments are plotted. (D) A total of 20 nM the control or CDC20 siRNA was transfected into HeLa cells. At 72 h posttransfection, cells were treated with 100 μg/ml of cycloheximide and harvested at 0, 2, 4, 6, or 8 h. Lysates were immunoblotted for PHF8, cyclin B1, CDC20, and actin, with bands quantitated as described above for panel C (I). The means and standard deviations of normalized PHF8 (II) and cyclin B1 (III) levels from triplicate experiments are plotted.