FIG. 7.
Mutation of threonines 447 and 451 does not affect the activity of AFX-A3. (A) Jurkat T cells were transfected by electroporation. Cells were subjected to electroporation with 10 μg of either pMT2-HA-AFX-A3, pMT2-HA-AFX-A3-T447A/T451A, or pMT2-HA-AFX-ΛDB; 4 μg of pCMV-p27Kip1-Luc reporter construct; and 4 μg of pCMV-LacZ in a total of 50 μg of DNA equalized with empty vector plasmid. Cells were analyzed for luciferase activity 48 h after transfection. The increases in luciferase activity are shown as fold induction over the activity in cells transfected with control plasmid alone. The results represent the averages of at least four independent experiments. The error bars represent the standard deviations of the values. Protein expression levels were regularly controlled by immunoblotting. (B) A14 cells were transfected with pEGFP in combination with 2 μg of either empty vector, pMT2-HA-AFX-A3, pMT2-HA-AFX-A3-T447A/T451A, or pMT2-HA-AFX-ΛDB. Cells were grown overnight with nocodazole (250 ng/ml). DNA profiles of GFP-positive cells were analyzed on a fluorescence-activated cell sorter. The absolute increase in percentage of A14 cells in the G0/G1 phase upon expression of the indicated proteins is presented. The error bars represent the standard deviations of the values. Protein expression levels were controlled by immunoblotting.