CARD11 aggregation and elevated NF-κB activity in patient primary B cells. (A) Expression of CARD11 and p65 visualized by immunofluorescence and confocal microscopy in fixed, purified naive B cells. The percentage of cells with visible CARD11 aggregates (marked by white arrowheads) or >50% p65 in the nucleus was quantified for each sample in by scoring >200 cells from multiple fields. Insets in bottom row show individual cells from the same field. Scoring data represent the mean ± standard deviation from two independent scorers. Bars: (top) 1 µm; (bottom) 10 µm. (B) Representative immunoblots of total naive B cell lysates prepared from normal donor controls (C1 and C2) and patients for proteins listed on the right of each blot. Arrowheads denote phospho–IKK-β (open) and phospho–IKK-α (closed). (A and B) Data are representative of three independent experiments. (C) Hierarchical cluster analysis of RNA-Seq data derived from resting B cells (pooled from six normal donors), activated B cells (3 h, 24 h), GC B cells (two donors), and plasma cells (PC) relative to patients, measured as reads per kilobase of exon model per million mapped reads (RPKM) from RNA-Seq analysis. Heat map projections of selected NF-κB target genes (listed at right) are shown, including NF-κB signature genes (top; Davis et al., 2001). (D) Comparison of digital gene expression (RPKM) for selected NF-κB target genes listed at left.