Regulation of TCF-1 and β-catenin levels and TCF/catenin signaling. (A) The wnt reporter transgene consists of 7 consensus TCF-binding sites upstream of a minimal promoter-TATA box, which drive the expression of β-galactosidase (β-gal). (B) Histograms show profiles reflecting β-gal activity in enriched populations of DN3 and DN4 thymocytes from BAT-GAL (gray line) and control mice (filled histograms). Small versus large DN3 cells were gated according to their forward and side scatter characteristics. (C) RAG-1-deficient mice were injected with anti-CD3ϵ mAb, and thymocytes were isolated before (0 hours) or 6 hours and 24 hours later. Total thymocyte lysates, corresponding to 10⁶ cells per lane, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred and subjected to Western blot analysis with the indicated antibodies. Molecular weight markers are indicated at right in kDa. (D) Quantitative RT-PCR analysis for β-catenin and Tcf-1 mRNA. RAG-1-deficient mice were injected with anti-CD3ϵ mAb, mRNA was isolated at the indicated time points and subjected to quantitative PCR (qPCR) analysis. Tcf-1 and β-catenin mRNA levels are indicated relative to that of TBP (TATA-binding protein) as described in “Materials and methods.” Bars show the mean ratio ± SD of triplicate determinations.