Induction of SPRY2 protein expression by CD40/BCR costimulation in B cells is ERK1/2 and TCL1 dependent. (A) SPRY2 protein expression in sorted WT B cells (pooled from 3 mice) stimulated with anti-CD40 Ab for 24 hours followed by costimulation with anti-IgM Ab for the indicated times. Freshly isolated splenic B cells before CD40 or BCR stimulations are shown in lane 1. The number beneath each lane shows the SPRY2 protein level normalized to the ACTIN level, as determined by densitometry. (B) SPRY1 and SPRY2 protein expression in WT B cells. Freshly isolated splenic B cells (pooled from 2 mice) before (−) or after stimulation (+) with anti-CD40 for 24 hours, followed by anti-IgM for 24 hours. Human embryonic kidney (HEK) 293T cells overexpressing SPRY1 or SPRY2 were used as positive controls (data not shown). (C) SPRY2 protein expression in WT B cells pooled from 2 mice costimulated as in panel B without (−) or with (+) U0126, a MEK1/2 inhibitor. (D) CD19-sorted human tonsil B cells were stimulated for the indicated times with phorbol 12-myristate 13-acetate and ionomycin, followed by Western blot. (E) Nalm-6 cells were infected with a retrovirus expressing a constitutively active form of MEK1 (CA-MEK1) or an empty vector control, followed by stable cell line establishment and Western blot. (F) ERK1/2 phosphorylation time course in WT (wt) and nonmalignant TCL1-tg (tg) splenic B cells costimulated by CD40/BCR without (−) or with (+) U0126. (G) SPRY2 protein expression in nonmalignant TCL1-tg (tg) or WT (wt) splenic B cells costimulated for 18 hours as in panel A. B cells were pooled from 2 TCL1-tg or 2 WT mouse spleens. The number beneath each lane shows the SPRY2 protein level normalized to the ACTIN level, as determined by densitometry. The mean and SD from this and a repeat experiment are plotted (right), normalized to the expression level for WT uninduced B cells. Data are representative of 2 (C-E), 3 (A,B,F), or 4 (G) independent experiments.