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Differential Expression of Functionally Glycosylated α-DG During T Cell Development.
(A) Thymi were harvested from 6- to 8-week-old C57BL/6 mice. Whole thymocyte suspensions were stained with lineage antibody cocktail (Lin, CD19/NK1.1/CD11c/TCRγδ), CD4, CD8, and IIH6 (for glycosylated α-DG). Lin− cells were gated and defined the major thymocyte subsets by CD4 and CD8 as DN (CD4−CD8−), DP (CD4+CD8+), CD4+, and CD8+ T cells (left). DN lymphocytes were further segregated into various subpopulations as CD44+CD25− DN1, CD44+CD25+ DN2, CD44−CD25+ DN3, and CD44−CD25− DN4. Glycosylated α-DG was detected with monoclonal antibody IIH6 (right). The data represented are from at least three independent experiments. Bold line, anti-glycosylated α-DG; shaded area, unstained. (B) Thymocytes were sorted into various subsets as indicated and lysed as described in Experimental Procedures. The protein extracts were subjected to SDS-PAGE followed by laminin binding (laminin) or by immunoblotting for β-DG (core DG protein) and β-actin (loading control) expression. (C) Detection of the messages for POMT1, LARGE1, LARGE2 by quantitative RT-qPCR: Complementary DNA (cDNA) was synthesized from total RNA of each of the cell types studied. For each cell type, POMT1, LARGE1, LARGE2 and Rps29 (normalization control) were specifically amplified, in triplicate, in the presence of SYBR green. The expression of POMT1, LARGE1 and LARGE2 are shown relative to that of the Rps29 in the same sample.