Figure 2b.

(C) Vβ8.1 and Vβ8.2 expression in sorted DN2/3 thymocytes, total thymocytes and sorted Vβ8.1/8.2 peripheral T cells from Vβ8.2^CD2/CD2, Vβ8.2^CD2/+ and wild-type mice. mRNA were extracted from each cell population and RT–PCR-amplified before subject to AflIII digestion as described in Materials and methods. Vβ8.1 cDNA remained as 450 bp full-length PCR product and Vβ8.2 cDNA was converted to a 390 bp fragment after AflIII digestion. Control reaction was carried out with a 400 bp Vβ8.2 genomic sequence containing the same AflIII site. Complete digestion of control DNA produced a 340 bp fragment. Size markers (M) are 100 bp DNA ladder. (D) Vβ8.2 and Vβ5.1 germline transcription in total thymocytes from Vβ8.2^CD2/CD2RAG2^−/− mice and RAG2^−/− mice. Germline transcription of Vβ8.2 and Vβ5.1 gene was determined by quantitative PCR and normalized to GAPDH expression. Normalized expression of each Vβ gene is plotted in relative to its expression in total thymocytes from RAG2^−/− mice. Results are the mean±s.e.m. of triplicates from one PCR as a representative of three independent PCR reactions. (E) Vβ8.2 rearrangement in sorted DN2/3 thymocytes from Vβ8.2^CD2/CD2 and wild-type mice. PCR analysis of genomic DNA in five-fold serial dilution was carried out with the Jβ2.7 reverse primer and the Vβ8.2 forward primer. Rearrangement products from Jβ2.1 and Jβ2.7 are indicated by the upper and lower boundaries of the bracket. DNA samples from thymocytes of RAG2−/− and LAT−/− mice were included as negative (N) and positive (P) controls, respectively. PCR amplification of the CD14 gene is used as a loading control shown at the bottom panel.