PMC

A) CH12.TRAF3^+/+ or CH12.TRAF3^−/− mouse B cells expressing hCD40 were stimulated with control (−) or anti-hCD40 (+) Ab-coated beads for 10 min. hCD40 was immunoprecipitated using the same beads. B) Quantitation by luminescence imaging of the results of 3 independent experiments, mean values ± SEM. *p<0.05.

A) A20.TRAF3^+/+ or A20.TRAF3^−/− mouse B cells were stimulated with anti-mCD40 Ab for 0, 5, 10, or 30 min. Western blots show pTAK1 and TRAF3, with actin as a loading control. B) Similar to panel A, except that A20.TRAF6^+/+ or A20.TRAF6^−/− mouse B cells were used. Western blots show pTAK1 and TRAF6, with actin as a loading control. These images were part of the same membrane, but the middle lanes were omitted here for clarity. Data shown are representative of at least four independent experiments.

CH12.hCD40 or CH12.hCD40LMP1 cells were treated with medium alone or IPTG for 18 h to induce DN-TAK1 prior to stimulation with anti-CD40 Abs, where anti-mCD40 Ab induced signaling by endogenous mCD40, and anti-hCD40 Ab induced signaling via either hCD40 (left) or hCD40LMP1 (right). After 72 h, samples were assayed for IgM production by hemolytic plaque assay. Data are presented as plaque-forming cells (Pfc, IgM-producing cells) per 10⁶ viable recovered cells, mean values ± SEM of replicate samples, and are representative of 3 independent experiments.

A) Mouse CH12.LX B cells were treated with DMSO or TAK1i for 30 min prior to stimulation with anti-mCD40 Ab for 0, 2, 5, 10, or 30 min. Western blots show pTAK1, pJNK, pIκBα, and total IκBα, with total JNK and actin as loading controls. B) Similar to panel A, except that CH12.hCD40LMP1 mouse B cells were stimulated with anti-hCD40 Ab for 0, 10, 30, or 60 min. Data shown are representative of three independent experiments.

A) WT mouse splenic B cells were treated with DMSO or TAK1i prior to stimulation with control insect cells (control i) or those expressing mCD154 (mCD154 i), as described in Methods. Cells were cultured for 24 h and supernatants were collected for IL-6 ELISAs. B) Similar to panel A, except that B cells were from a mCD40LMP1 Tg mouse. C) Similar to panel A, except that human B cells were stimulated. D) Similar to panel A, except that CH12.hCD40LMP1 mouse B cells were treated with medium alone (control) or IPTG to induce DN-TAK1 prior to stimulation. Data in all panels are mean values ± SEM of triplicate samples, and are representative of at least two independent experiments.

A) WT mouse splenic B cells were treated with DMSO or TAK1i for 30 min prior to stimulation with anti-mCD40 Ab for 0, 5, 10, or 30 min. Western blots show protein levels for pJNK, with actin as a loading control. B) Similar to panel A, except that splenic B cells from a mCD40LMP1 Tg mouse were stimulated for 0, 10, 30, or 60 min. C) Mouse CH12.LX B cells were treated with DMSO or TAK1i for 30 min prior to stimulation with anti-mCD40 Ab for 0, 2, 4, or 6 h. Western blots show p-c-jun, with actin as a loading control. D) Similar to panel C, except that CH12.hCD40LMP1 mouse B cells were stimulated with anti-hCD40 Ab. Data shown are representative of at least two independent experiments. E) CH12.hCD40LMP1 cells were treated with DMSO or JNKi prior to stimulation with control insect cells (control i) or cells expressing either mCD154 (mCD154 i) or hCD154 (hCD154 i). Cells were cultured for 24 h and supernatants were collected for IL-6 ELISA assay. Data are mean values ± SEM of triplicate samples from two independent experiments.