Extended Data Fig. 2 |. pHEL signaling is sensitive to ED but not affinity and is potent in both follicular and MZ B cells.

a-c. MD4 splenocytes and lymphocytes were stimulated as in Main Fig. 2A,B but assessed for intra-cellular pS6 over a time course. Graphs (left) and histograms (middle) depict pS6 in B220+ cells from a single time course and is representative of 2 independent experiments. Graphs (right) depict B cell pS6 MFI after 20 min. Data are pooled from n = 5 (HELD), n = 6 (HELT) experiments. C. Graph depicts pHELT and pHELD data from A, B graphed together for comparison. A-C were compared by two-tailed paired parametric T-test, with mean depicted. d. As in 2 A, B but comparing pErk in stimulated B220+ splenocytes and lymph node cells separately at 20 mins. Histograms are representative of 2 independent experiments. e-g. As in 2 A, but splenocytes were stained with B220, CD21, and CD23 to identify pErk expression in subsets. Representative histograms show pErk in gated subsets following sHEL-WT 1 μg/ml, 1pM pHELT-HD (ED 256), or PMA stimulation. F. Quantification and mean of %pErk+ cells from three independent experiments (G). Groups were compared by one-way ANOVA. Data in E-G are representative of 8 independent experiments. h, i. Pooled MD4 splenocytes and lymphocytes were loaded with Indo-1 calcium indicator dye, stained with surface markers to detect B220+ subsets (H) as in S2E, and stimulated with anti-IgM (10 μg/ml), sHELD (1 μg/ml), or pHELD-HD (1pM). Calcium entry was assessed by flow cytometry for >= 3 minutes. MFI of bound/unbound Indo-1 fluorescence over time is plotted (I). Data are representative of 4 independent experiments. J. As in Main Fig. 3a,b except CD23+ B cells stimulated with 10, 1, or 0.1pM doses of either pHELT-HD (ED 256) or pHELT (ED 3). Data are representative of 4 independent experiments.