Supplementary Figure 5: Related to Fig. 5. Ikaros controls anergy induction upon chronic antigen exposure.
From: Ikaros prevents autoimmunity by controlling anergy and Toll-like receptor signaling in B cells
a, Intracellular BCR signaling. The phosphorylation (p-) status of signal transducers downstream of the BCR was determined in lymph node FO B cells of MD4 BCR-tg Ikzf1B+ (purple shaded or dashed line) and MD4 BCR-tg Ikzf1B– (purple line) before (unstimulated (unst.), dashed line) and after 5 min of anti-IgM addition or 15 min of HEL stimulation. Flow cytometric analysis was performed with antibodies specific for p-SYK (p-Tyr525/526), p-BLNK (p-Tyr84), p-PLCγ2 (p-Tyr759), p-ERK1/2 (p-Thr202/204), p-AKT (p-Ser473) and p-S6 (p-Ser235/236). b, Measurement of intracellular Ca2+ fluxes in HEL-stimulated CD43– FO B cells from lymph nodes of MD4 BCR-tg Ikzf1B+ or MD4 BCR-tg Ikzf1B–mice, which were either not pre-treated (naïve), were injected with HEL antigen for 1 day or additionally carried the ML5 transgene. c, Evaluation of the Ikzf1 deletion frequency by intracellular Ikaros staining of transitional T1 B cells and mature B cells (CD19+CD5–CD138–CD93–CD23+IgD+) from the spleen of MD4 BCR-tg Ikzf1B+ (purple shaded) and MD4 BCR-tg Ikzf1B– (purple line) mice lacking (upper row) or containing the ML5 transgene (lower row). d, HEL binding to the MD4 BCR receptor at 1, 4 and 20 hours after HEL injection into MD4 BCR-tg Ikzf1B– and control MD4 BCR-tg Ikzf1B+ mice, as shown by flow cytometric analysis of lymph node B cells (CD19+CD138–) with an anti-HEL antibody (HyHEL9). Data in a, c and d are representative for 3, 4 and 2 independent experiments, respectively. Data in b are representative of experiments analyzed in Fig. 5a, c, e.
