(A) Transfection of Foxp3-V5 into TSA cells repressed expression of the Skp2 gene. The mRNA levels of Foxp3, Skp2, and p27 were measured by real-time PCR for the vector control cells, polyclonal Foxp3-V5 transfectants (CL30), and 2 stable Foxp3-V5 transfectant clones, CL302 and CL305. Data shown are relative amounts of transcripts after normalizing against the amounts of total RNA based on the levels of Hprt mRNA. The means of the vector group are artificially defined as 1.0. Data shown are means ± SD of 3 independent experiments. (B) Foxp3 reduces Skp2 with a corresponding increase in p27. Lysates of Foxp3-V5 or vector-transfected TSA cell lines were analyzed by Western blot using SKP2, p27, β-actin, and anti-V5 (which recognize V5-tagged Foxp3) antibodies. (C) Foxp3 increased stability of p27. Vector or Foxp3-V5 transfectants were treated with cycloheximide (CHX, 100 μM) for the indicated intervals. Cells were collected and p27 protein levels were detected by Western blot. The upper panel shows representative experiments while the lower panel shows the decay of p27, using time 0 as 1.0. Protein loading equivalence was assessed by the expression of β-actin. The relative intensity of bands was measured relative to their respective β-actin bands using BandScan software (version 4.3; Glyco). (D) Polyubiquitination of the p27 in vector or Foxp3 transfectants. Vector or Foxp3-V5–transfected TSA cell lines were either left untreated or treated with proteasome inhibitor MG132 (40 μM) for 3 hours. Equal aliquot of the cellular lysates were immunoprecipitated with anti-p27 antibodies. The precipitates were separated by SDS-PAGE and transferred into nitrocellulose membrane, which were then autoclaved in water for 30 minutes and blotted with HRP-conjugated anti-polyubiquitin antibody Ub P4D1 (33).