Figure 4

Behaviors of Baf57^Int and Baf57^ΔR. (A) Western blot examining Brg/Brahma-associated factor (BAF)57 expression in total thymocytes (lanes 1 to 5, and lane 10) and purified CD4 cells (lanes 6 to 9). Samples in lanes 8 to 10 were from mice exposed to TAM. The genotype of the mice are indicated, where F, ΔR and Flp denote Baf57^F, Baf57^ΔR and R26^FlpoER1, respectively. The bracket at the left indicates the three BAF57 mutant proteins expressed from Baf57^F after deletion of floxed sequence. At least three mice of each genotype were analyzed. Shown is a representative experiment. (B) Reverse transcriptase (RT)-PCR examining BAF57 transcripts in CD4 cells expressed by the wild-type (WT) allele (lanes 1 and 3) or the mutant allele lacking exons 2 and 3 (lanes 2 and 4); the cells were isolated from Baf57^+/ΔR; CD4-Cre; R26^FlpoER1 and Baf57^F/ΔR; CD4-Cre; R26^FlpoER1 mice, respectively. RT-PCR primers targeted exons 1 and 7 (lanes 1 and 2) or 1 and 5 (lanes 3 and 4). The latter primer set yielded amplicons A to C (lanes 3 and 4, diagrammed at the bottom), which were re- amplified with a nested primer set, gel-purified, and digested with EcoN1 to verify their identities (lanes 5 to 10). There was also a nonspecific amplicon (about 310 bp, lane 3) which was not re-amplifiable (not shown). Two mice of each genotype were analyzed. Shown is a representative experiment. (C). Flow cytometric assays monitoring green fluorescent protein (GFP) expression in peripheral blood CD4, CD8 and B cells at various times after tamoxifen (TAM) injection. Three mice of each genotype were analyzed. (D) Summary of the reversion efficiencies at day 10 in CD4, CD8, and B cells, as measured by the fraction of cells that had lost GFP. Each symbol represents an individual mouse.