Figure 3 - Foxo1 functions 'downstream' of GADD45a to increase Rag1 transcription in AMuLV-transformed pro–B cells.

(a) Immunoblot analysis of Foxo1, Foxo3A and Foxo4 in AMuLV-transformed B cells infected with retroviruses expressing shRNA (top) or mRNA (bottom). For shRNA knockdown, numbers below lanes indicate the amount of Foxo1 or Foxo3a, after normalization to -actin (loading control), relative to that in cells infected with empty vector (set as 100). Foxo4 protein is undetectable in the absence of overexpression. For protein overexpression, cells infected with retrovirus-expressing Foxo protein are compared with cells infected with empty vector retrovirus, with -actin as a loading control. (b,c) GFP expression in ER-GADD45a-expressing cells infected with retrovirus encoding shRNA (b) or mRNA (c) and treated with tamoxifen for 24 h, then labeled with anti-hCD2 or anti-Thy-1.1 and gated for infected cells (solid lines) or uninfected cells (filled histograms). Numbers above bracketed lines indicate percent GFP⁺ cells (top number) and the mean fluorescence intensity (bottom number). (d) Immunoblot analysis of Foxo1 in AMuLV-transformed B cells infected with empty virus retrovirus or retrovirus expressing GADD45a. Numbers below lanes indicate the ratio of Foxo1 to -actin. All data are representative of three independent experiments.