Evidence for the physiologic role of incomplete forms of TCRβ in thymocytes. (A) 293 cells were transfected with the indicated constructs. At 48 hours after transfection the cells were fixed and stained with propidium iodide. Cells were than separated by analysis to different populations according to their GFP expression (i-iii,vii,viii). Each population was then further analyzed for its cell-cycle distribution. (B) Twenty-four hours after transfection with Zap70-YFP or EGFP-iJC, the protein extracts of Jurkat cells were loaded onto 10% SDS-PAGE gels. Immunoblot analyses were preformed with anti-Zap70 antibody and after stripping with anti-GFP (i). The 2 lanes shown were cut from the same blot. The calculated molecule/cell numbers of the rZap70-YFP or EGFP-iJC proteins are displayed in the table (ii). (C) The cell lines (S49.1 or SCID, 5 × 10⁷ cells/sample) (i) or thymocytes (WT and TCR^−/−, 2 × 10⁸ cells/sample) (ii) were lysed, and TCRβ was immunoprecipitated by beads cross-linked to anti-TCRβ antibodies. Samples were separated on SDS-PAGE and probed with anti-TCRβ antibodies. The lack of corresponding bands in lymphocytes from SCID and TCR^−/− cells show the specificity of the antibodies. Analysis of extracts from thymocytes from either untreated mice, cells from mice irradiated at 6 Gy (iii) or thymocytes at 1 or 4 weeks of age (iv) shows the presence of low-molecular-weight forms reactive with the antibody. The blot was cut (dotted line) and redeveloped to allow intensification of the lower molecular-weight bands (v). Peptides were isolated.