Constitutive binding of ZAP-70 to BCR kinases and ribosomal proteins. (A) PCA of protein abundance, representing the variability between anti–ZAP-70 pull-down, with or without anti-IgM stimulation for 20 minutes, and IgG isotype control. Each dot represents 1 technical replicate with an individual pull-down of ZAP-70 or IgG. (B) Heat map of proteins annotated in the Kyoto Encyclopedia of Genes and Genomes “BCR signaling pathway,” identified by mass spectrometry as ZAP-70 binding partners. Red and blue indicate relatively high and low protein abundance, respectively. Each condition analyzed depicts 3 (unstimulated) or 4 (IgM-activated) technical replicates. Proteins were ranked from left to right by Log2(FC) ratio comparing the IgM-activated group with the unstimulated group. (C) Volcano plot showing proteins bound to ZAP-70 [Log2(FC) > 0] vs IgG isotype control [Log2(FC), < 0] in unstimulated CLL cells. (D) Gene set enrichment analysis was applied to identify pathways with which ZAP-70–associated proteins are associated in unstimulated CLL cells. Pathways are listed in order of Normalized Enrichment Scores in columns (top x-axis). FDR q-values for each pathway set are indicated by the red dotted line (bottom x-axis). Colors of the columns represent different subgrouped cell functions. (E) Volcano plot showing proteins bound to ZAP-70 [Log2(FC) > 0] vs IgG isotype control [Log2(FC) < 0] in anti-IgM–activated CLL cells. (F) Gene set enrichment analysis to identify pathways associated with ZAP-70 in anti-IgM–activated CLL cells. (G) Volcano plot showing proteins differentially bound to ZAP-70 in unstimulated [Log2(FC) < 0] vs anti-IgM–activated [Log2(FC) > 0] states. (H) Gene set enrichment analysis to identify pathways associated with ZAP-70 after BCR activation with an anti-IgM.