Fbw7γ regulates KLF13 in human T cells. (A) Total RNA was prepared from human T cells activated with anti-CD3 plus anti-CD28 Abs for 0 (black bar), 1 (dark gray bar), 2 (medium gray bar), 3 (light gray bar), or 5 (open bar) days. The mRNA level of each Fbw7 isoform was determined by RT-PCR and normalized to the level in day 0 resting T cells. (B-C) Human T cells were transfected with control siRNA or siRNA for each isoform of Fbw7. After 24 hours, the cells were analyzed by immunoblot assay with the use of anti-KLF13 Ab (B) or by RT-PCR (C). (D) Relative expression levels of human and mouse Fbw7 were determined by RT-PCR and normalized to HPRT expression. (E) HEK293T cells (lanes 1-2) were transiently transfected with human KLF13 (lane 1) or mouse V5-tagged KLF13 (lane 2) and HA-tagged ubiquitin (Ub). The cell lysates were immunoprecipitated (IP) with anti-KLF13 Ab for Western blot analysis with anti-HA Ab or anti-KLF13 Ab. ED1 cells (lanes 3-10) were transiently transfected with plasmids expressing human (lanes 3-6) or mouse V5-tagged KLF13 (lanes 7-10), Flag-tagged Fbw7α (lanes 4,8), Fbw7β (lanes 5,9), Fbw7γ (lanes 6,10) and HA-tagged Ub (all lanes). The cell lysates were IP with anti-V5 Ab for Western blot analysis with anti-HA Ab or V5 Ab. Data are mean ± SD.