FIG. 5.

Characterization of thymocytes lacking mSin3A. (A) Genomic PCR analysis of thymocyte DNA derived from msin3A^L/+ and msin3A^L/L thymocytes with and without lckCre. The position of wild-type (WT), targeted (LoxP), and deleted (Δexon 2) alleles are shown (upper panel). (B) Western blot analysis of protein extracts derived from thymocytes with the indicated genotype. Antiserum which recognizes the PAH2 domain (amino acids 304 to 380) was used to detect mSin3A protein. The white arrowhead indicates the position of the truncated mSin3A protein. (C) The number of thymocytes harvested from individual animals with the indicated genotype was determined using a hemocytometer. The average cellularity was calculated using four mice of each genotype from three independent experiments. (D) Single cell populations of thymocytes harvested from mice with the indicated genotypes were stained with FITC-conjugated anti-CD25, anti-CD8, anti-gamma delta TCR, PE-conjugated anti-CD44, anti-CD4, and biotinylated-anti-CD3ɛ, followed by streptavidin tricolor. A representative flow cytometric histogram from 8-week-old mice is shown. (E) The average cellularity of each thymocyte subset was determined, using flow cytometric analysis and total thymic cellularity, from three independent experiments. (F) Splenocytes derived from the genotypes indicated were stimulated to proliferate ex vivo by addition of anti-CD3 and anti-CD28 antibodies. The cells were allowed to divide for 48 h and then pulsed with ³[H]thymidine for 24 h prior to harvesting. A representative histogram of the average ³[H]thymidine incorporated, normalized for the number of starting T cells, is shown. (G) Splenocytes were labeled with CFSE for 5 min at room temperature. The cells were then stimulated to proliferate as for panel B and then analyzed at 48 and 96 h by flow cytometry to assess division.