Spi-B is expressed in human B-cell subsets, but not in plasma cells. Equal numbers of CD19⁺ B cells of (A) peripheral blood or (B) tonsil were sorted into subpopulations as indicated by gating and analyzed by immunoblotting. Actin levels were determined as loading control. One representative experiment of 3 is shown. (C,D) CD19⁺ B cells purified from peripheral blood were cocultured under plasma cell promoting conditions. (C) Naive (left panel) or memory (right panel) B cells were cultured for 7 days and analyzed for CD20 and CD38 expression by flow cytometry. Numbers in the quadrants indicate percentages of cells. For immunoblotting, cells were collected at the indicated time points and analyzed for Spi-B and BLIMP1 expression. Actin levels were determined to ensure equal loading of the samples. (D) Memory B cells were cultured for the time periods indicated, and expression levels were assessed by quantitative real-time PCR. Mean plus or minus SD values of PCR duplicates are shown. One representative experiment of 2 is shown.