A regulatory subunit SCN4B is critical in positive selection. (a, b, c) Left: Flow cytometry of thymocyte differentiation induced in reaggregate culture. The reaggregate cultures in a,b,c were set up as described in Fig. 3a,b,c respectively, with treatment of SCN4B-Ig or unrelated Ctrl-Ig (Ceacam4-Ig) fusion protein. Right: Quantification of percent subpopulation; each symbol represents a reaggregate culture. All P values were calculated by two-tailed Mann-Whitney test. (a) gp250 induced AND CD4SP positive selection with SCN4B-Ig or Ctrl-Ig (10 μg; mean ± s.d.; n = 6 in three experiments; *P = 0.0022). (b) Positive selection of AND CD4SP T cells induced by IFN-γ–treated ANV41.2 cells with 10 μg SCN4B-Ig or Ctrl-Ig (mean ± s.d.; n = 7 in three experiments; *P = 0.0041). (c) Positive selection of polyclonal pre-selected B6.K DP thymocytes with 1 μg SCN4B-Ig or Ctrl-Ig (mean ± s.d.; n = 9 in three experiments; *P = 0.0018). (d) Fura-2 Ca2+ flux of pre-selected AND.Rag1−/−H-2d DP thymocytes stimulated with gp250–I-Ek with SCN4B-Ig or Ctrl-Ig (1 μg). (e) Summary of peak and mean value of intracellular Ca2+ concentration from 7.5 to 15 min of two experiments each involving 50 cells (*P < 0.0001; **P < 0.0001)