NF-κB is activated through the canonical pathway in LN-derived CLL cells. (A) Genes in the NF-κB signature that were most significantly enriched in the LN as identified by GSEA (leading edge genes) are depicted in a heat map for the 12 patients having contributed cells from all 3 compartments. (B) The NF-κB score was computed as the average of the mRNA expression level of the leading edge genes for each sample. Shown is the ratio of the BCR score in BM (n = 19) or LN (n = 17) relative to the score of the matched PB sample. Comparison between BM and LN was by Student t test. The sample with the exceptionally high score in the BM was obtained from CLL_C10. (C) Western blot of nuclear lysates from purified CLL cells; JUNB expression was normalized to TBP and the ratio between LN- and PB-derived cells is shown. Western blots of cytoplasmic protein fractions from purified CLL cells were probed for phosphorylated IκBα (p-IκBα, Ser 32-36; D) and for total IκBα (E). IκBα and p-IκBα expression quantified by densitometry was normalized to γ-tubulin and the ratio between LN- and PB-derived cells is shown. (F) Pearson correlation between IκBα expression and the NF-κB gene expression score.