Gfi1b is expressed during a small window of early T-cell development. (A) Thymocytes from adult wt or wt/KI (Gfi1b^+/GFP) mice were stained for the surface markers CD4 and CD8 and gated for different stages of T-cell development as described previously.⁹  GFP fluorescence of the respective subpopulations is shown in representative histograms (right). (B) Because only in the double-negative (DN) population GFP-positive cells were present, thymocytes from adult wt (▪) or wt/KI (Gfi1b^+/GFP) mice (⊡) were stained for CD25 and CD44 expression and lineage-negative (Lin⁻) cells were electronically gated out as shown (left). The GFP expression of the respective gated cell population is shown in representative histograms (right). (C) Representative diagram showing the expression of GFP during early T-cell development, shown as the difference of the geometric mean fluorescence intensities between wt and wt/KI (Gfi1b^+/GFP) cells (left). Quantitative RT-PCR (real-time analysis) showing mRNA levels for Gfi1 and Gfi1b during early T-cell development normalized to the respective expression levels in DN1-preDN2 cells (right). (D) Western blot analysis of Gfi1b protein showing the decrease of Gfi1b during early B-cell development (left) and quantitative mRNA analysis for Gfi1 and Gfi1b during early B-cell development and in early erythroid cells (right). Error bars represent the standard deviation of triplicate measurements.