FIG. 7.

The phosphorylation of FKHRL1 at the three regulatory sites is required to prevent FKHRL1-induced cell cycle arrest and apoptosis. (A) CCL39 fibroblasts were transfected with the various phosphorylation site mutants of FKHRL1, starved for 24 h, and stimulated for 24 h with 20% FCS in the presence of BrdU. Transfected cells and the incorporation of BrdU were identified by immunofluorescence with an anti-FKHRL1 antibody and an anti-BrdU antibody, respectively. Data are the means and variances of two independent experiments. CTL, control; TM, triple mutant of FKHRL1 (T32A/S253A/S315A). (B) HEK 293T cells were transfected with the various FKHRL1 phosphorylation site mutants and stimulated for 24 h with IGF-I. Immunoblot analyses were performed with an antibody directed against the cleaved product of PARP, a control antibody to a protein whose levels remain constant (MKK1), and an antibody to HA. TM, triple mutant of FKHRL1 (T32A/S253A/S315A) .