Extended Data Fig. 5. Signaling in DP thymocytes in Lck variant mice.

(a) Thymocytes of indicated Lck variants OT-I Rag2^KO/KO β2m^KO/KO mice were activated ex vivo with T2-Kb cells loaded with OVA peptide or APLs with decreasing affinity (OVA > T4 > E4 > E1) overnight and analyzed for the CD69 expression by flow cytometry. Mean values + /- s.e.m. are shown. n = 6 independent experiments/mice for Lck^WT vs. Lck^CA (upper row), n = 3 independent experiments/mice for Lck^WT vs. Lck^CA/KR (bottom row). Differences in the EC50 of the fitted non-linear regression curves were tested using extra sum-of-squares F test. F, p, and fitted EC50 values are shown. (b) Fetal thymic organ cultures from indicated Lck variant OT-I Rag2^KO/KO β2m^KO/KO mice were stimulated with OVA peptide (20 µM) or its APLs decreasing affinity (Q4R7 > Q4H7; 2 µM) and analyzed by flow cytometry after 7 days. Individual mice and means are shown. n = 13 for Lck^WT/WT no peptide and Q4H7 in 5 independent experiments, n = 14 for Lck^WT/WT Q4R7in 5 independent experiments, n = 4 for Lck^WT/WT OVA in 2 independent experiments, n = 13 for Lck^CA/CA no peptide in 5 independent experiments, n = 8 for Lck^CA/CA Q4H7 in 3 independent experiments, n = 14 for Lck^CA/CA Q4R7 in 5 independent experiments, n = 5 for Lck^CACA OVA in 2 independent experiments, n = 10 for Lck^KO/KO no peptide in 4 independent experiments, n = 8 for Lck^KO/KO Q4H7 in 3 independent experiments, n = 9 for Lck^KO/KO Q4R7 in 3 independent experiments, n = 6 for Lck^KO/KO OVA in 2 independent experiments. Statistical significance was calculated using a Mann-Whitney test.

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