The Lck unique domain is required for T-cell development. (A) Schematic showing structural domains of WT (top) and FU-Lck (bottom) proteins. (B) Western blot analysis showing relative expression levels of Lck and FU-Lck proteins in thymocyte lysates from mice from lines 1 (L1) and 2 (L2) of Lck^tg mice and lines 1 and 3 (L3) of FU-Lck^tg mice. A V5 mAb was used to detect transgenic Lck, which was tagged with the V5 epitope, while ERK reprobes served as loading controls. (C) Dot plots showing distribution of T-cell subsets in thymi from 7-week-old WT, Lck^−/−, Lck^tg/Lck^−/−, and FU-Lck^tg/Lck^−/− mice as defined by surface CD4 and CD8 expression and FACS analysis. Values within dot plots represent proportions of cells within CD4 SP, DP, and CD8 SP gates. Dot plots represent staining from 1 of at least 8 mice of each genotype. (D) FACS dot plots showing distribution of DN populations in mice thymi. γδTCR⁻CD4⁻CD8⁻ cells were subdivided into DN1 to DN4 populations by expression of CD44 and CD25: DN1 = CD44⁺CD25⁻; DN2 = CD44⁺CD25⁺; DN3 = CD44⁻CD25⁺; and DN4 = CD44⁻CD25⁻. Values represent the proportion of cells in each DN population, and dot plots represent analysis of 1 of at least 5 mice of each genotype. (E) Histograms showing FACS analysis of γδTCR expression in CD4⁻CD8⁻ DN populations from mouse thymi. Values represent proportions of γδTCR-positive and TCR-negative cells as defined by the respective gates. Data are representative of 1 of 5 mice analyzed of each genotype. (F) Histograms showing DP thymocyte expression of surface αβTCR (left side) and CD5 (right side). Filled histograms represent levels of expression of αβTCR or CD5 in WT DP cells; fine black line overlays represent Lck^−/− levels; bold black overlays represent Lck^tg/Lck^−/− levels; and bold gray lines represent FU-Lck^tg/Lck^−/− levels. Data are representative of at least 5 mice of each genotype.