ASK1 enhances apoptosis during the generation of short-lived plasma cells. (A) ASK1 is activated during the generation of short-lived plasma cells. Splenic B cells treated with LPS (2 μg/mL) or the combination of IL-21 (100 ng/mL), αCD40 (5 μg/mL), and αIgM (10 μg/mL) for various periods were subjected to immunoblotting with the indicated antibodies. Actin was used as the loading control. Phosphorylated ASK1 is denoted as p-ASK1. (B) Ectopic expression of ASK1 promotes apoptosis of short-lived plasma cells. Murine splenic B220⁺ B cells stimulated with IL-21, αCD40, and αIgM were transduced with retroviral vectors producing either WT or CA ASK1 or with a mock virus (pGC) followed by annexin V staining and FACS to measure apoptosis in the yfp⁺B220^lowCD138⁺ gate at 2 days after transduction. The diagram above the histograms shows the linear domain map of ASK1. (C) Plasma cells gated from the stimulated Ask1 KO splenic B-cell culture show decreased apoptosis. B220⁺ splenic B cells isolated from WT or Ask1 KO mice were cultured with LPS or the combination of IL-21, αCD40, and αIgM for various periods. The extent of apoptosis of B220^lowCD138⁺ plasma cells was analyzed by annexin V⁺ staining. (D-E) Increased NP-specific antibody-secreting cells were detected in the spleen (D) or bone marrow (E) of Ask1 KO mice. Splenocytes or bone marrow cells from WT and Ask1 KO mice harvested at 1 week (D) and 6 weeks (E) after NP-Ficoll (D) or NP–keyhole limpet hemocyanin (E) immunization were used to quantify NP-specific IgM-secreting (D) or IgG-secreting (E) cells by ELISPOT analysis. Each closed or open circle represents an individual WT or Ask1 KO mouse (D: n = 5; E: n = 4). *P < .05.