Figure 4.

Intracellular staining of Vβ3 in Vβ8⁺ T cells from AND/MBP mice. FACS analysis of lymphocytes and thymocytes harvested from either double transgenic AND/MBP mice or single transgenic MBP mice that were stained with antibodies against CD4, Vβ3, or Vβ8. The histograms show the level of cell surface Vβ3 (thin line) and intracellular Vβ3 staining (thick line) on electronically gated CD4⁺, Vβ8⁺ T cells or on total thymocytes. Vβ3 staining of Vβ8⁺ (A) T cells and (C) thymocytes from AND/MBP mice can only be seen if cells are permeabilized before the addition of the Vβ3 antibody. The specificity of the intracellular staining of the AND TCRβ chain is confirmed by carrying out the same staining procedure on (B) T cells and (D) thymocytes from MBP TCR-only transgenic mice. Sorted T cells from AND (E), MBP (F), and Vβ8⁺ T cells from AND/MBP (G) transgenic mice were pulse labeled for 30 min with [³⁵S]methionine. The TCR α and β proteins were immunoprecipitated from Triton X-100 solubilized lysates with anti-Vβ8 (mAb F23.2), anti-Vβ3 (mAb KJ25), anti-Vα11 (mAb RR8–1), anti-Cβ (mAb H57), or control anti-HLA-DM (mAb MaP.DMB/c) and analyzed by SDS/PAGE (11%). The positions of the individual TCR β and α chains are indicated.