Noxa is induced in activated B cells. (A) Representative FACS plots of activated B cells. Gated is for B220⁺ cells. Top panels: B cells isolated directly ex vivo from (left) naive or (right) mice immunized 12 days prior with 50 ug of TNP-KLH (30:1) in alum. Bottom panels: in vitro cultured, purified B cells after 4 days of stimulation with anti-IgM and LPS in the (left) absence or (right) presence of 20 ng/mL IL-4. (B) Expression profiling by RT-MLPA of purified mouse B cells after in vitro stimulation. Cells were stimulated with anti-IgM/LPS alone (−IL4), or in combination with IL-4 (+IL4). Gene induction of pro and antiapoptotic molecules is represented in a heat-map after log2 transformation of expression levels (scale −2.5-2.5), relative to averaged values of unstimulated cells at day 0 (n = 3 per genotype. Shown is one of at least 3 experiments). (C) RT-PCR analysis for Noxa, Bim, Mcl-1, and 18S ribosomal RNA of anti-IgM/LPS (−IL4) and anti-IgM/LPS/IL-4 (+IL4) stimulated B cells. (D) Western blot analysis of total purified B cells after anti-IgM/LPS (−IL4) and anti-IgM/LPS/IL-4 (+IL4) stimulation. A representative result of 3 independent experiments is shown. (E) Expression profiling by RT-MLPA of cell sorted FB (B220⁺IgM⁺IgD⁺CD21/35⁺), MZB (B220⁺IgM⁺IgD⁻CD21/35^bright), and GC (B220⁺CD38^dimGL7^bright) B cells of PE immunized mice, 12 days after immunization (n = 3 per genotype). Shown is 1 of 3 independent experiments. Mean and SEM are shown.