FIGURE 5.

Az2 stimulates ODC but not cyclin D1, Aurora-A, and ΔNp73 degradation and can be stably expressed in trypanosome ODC-expressing cells. A, HEK-293T cells were cotransfected with constructs encoding FLAG-tagged (f) Az2, ODC, Aurora-A, cyclin D1, and ΔNp73. Cycloheximide (CHX) was added to the growth medium 24 h post-transfection, cellular extracts were prepared at the indicated times, and the amounts of the individual tested proteins were determined by Western blot analysis using anti-FLAG antibodies. The data presented are representative of three repetitions. The asterisk denotes a nonspecific band. B, HEK-293T cells stably expressing trypanosome ODC (Tryp.ODC) were transfected with Az1 or Az2-expressing constructs. Focuses were initially selected with 1 mg/ml puromycin (Puro), followed by amplification of the expression unit by increasing puromycin to 5, and 20 mg/ml. Cellular extracts were prepared, and the expression levels of trypanosome ODC, Az1, and Az2 were determined by Western blot analysis. C, the growth rate of cells expressing trypanosome ODC and Az2 was compared with that of control cells transfected with two empty vectors by daily counting of cells as described under “Experimental Procedures.” *, significant difference from control cells using Student's t test (p < 0.02).