Figure 4: SH3–GUK regulation is essential for B-cell physiology mediated by CARMA1.
(a) Peritoneal IgM+CD5+ B-1 cells and splenic follicular IgMloIgDhi B-2 and CD21hiCD23lo marginal zone B cells from 8-week-old WT, Carma1L815P and Carma1–/– mice were analysed by flow cytometry. Data are representative of four independent experiments. (b) Basal serum immunoglobulin levels in 8-week-old WT, Carma1L815P and Carma1–/– mice were analysed. Each circle represents an individual mouse; error bars indicate the s.d. *P<0.01 and **P<0.001 (Student’s t-test). (c) Purified splenic B cells from WT, Carma1L815P and Carma1–/– mice were stimulated with the indicated amount of anti-IgM F(ab′)2 or 1 μg ml−1 of LPS for 48 h. Proliferation was measured by [3H]thymidine uptake. Data represent the mean±s.d. of triplicate cultures. (d–g) Purified splenic B cells from WT, Carma1L815P and Carma1–/– mice were stimulated with anti-IgM F(ab′)2 (10 μg ml−1) or with PMA (10 ng ml−1) plus calcium ionophore (1 μM; P/I) for the indicated times. Cell lysates were analysed by immunoblotting of actin (d), total (d,e) and phosphorylated IκBα (e) and total and phosphorylated JNK, ERK and p38 (g). (f) The electrophoretic mobility shift assay. Nuclear extracts were prepared from purified WT, Carma1L815P and Carma1–/– splenic B cells stimulated for 8 h with anti-IgM F(ab′)2 and P/I. Electromobility shift assays were performed using DIG-labelled oligonucleotide probes containing consensus NF-κB-binding sites. Results are representative of more than three independent experiments.
