Lethally irradiated C57BL6/J (CD45.1+) mice were reconstituted with lineage depleted Themis1−/− bone marrow cells (CD45.2+) that had been infected with a bicistronic EGFP expressing retroviral vector (Empty vector) or the same vector encoding either wild type Themis1 cDNA (WT) or mutant Themis1 proteins: Themis-ΔPRR (PRR), Themis-ΔNLS (NLS) or Themis-ΔCys (Cys1/2). Eight weeks post-reconstitution, bone marrow chimeras sacrificed and thymocytes and splenocytes were analysed by flow cytometry. a. Intracellular staining for Themis1 in CD45.2+ EGFP+ CD4+CD8+ (DP) thymocytes from bone marrow chimeric mice. Grey histograms represent Themis1 expression in EGFP− DP thymocytes. Green histograms represent expression of Themis1 in EGFP+ DP thymocytes, expressing the indicated Themis1 proteins. b. Representative flow cytometry analysis of CD45.2+ EGFP+ thymocytes from bone marrow chimeras. Upper plots show CD4 versus CD8 profiles, middle histograms show percent of TCRβ+ cells. Bottom plots show percent of mature CD45.2+ EGFP+ TCRβhiCD24lo thymocytes. Plots in right column show Themis1+/+ thymocytes infected with empty retroviral vector for reference. c. Percentage of mature (TCRβhiCD24lo) CD45.2+ EGFP+ CD4-SP and CD8-SP thymocytes in the indicated bone marrow chimeras (n=6 each). *p ≤0.05, N.S., not significant (Two-tailed T-test, unequal variance) All comparisons are to vector only.