BCR activation induces binding of ZAP-70 to ribosomal proteins and is associated with enhanced protein biosynthesis. (A) Venn diagram comparing ZAP-70–binding proteins identified by mass spectrometry (MS) in anti-IgM–activated (20 minutes) primary CLL cells and BJAB cells [Log2(FC) > 1; adjusted P < .01]. (B) Gene set enrichment analysis GO terms extracted from the 147 unique proteins, identified in (A), from primary CLL cells and BJAB cells. GO signatures are listed in order of Normalized Enrichment Scores in columns (top x-axis). FDR q-values for each signature set are indicated by the red dotted line (bottom x-axis). Colors of the columns represent different subgrouped cell functions. (C) The 147 unique proteins from (A) were plotted by Log2(FC) (x-axis, identified in primary CLL cells by MS; y-axis, identified in BJAB cells). Colors indicate the subgrouped functions of the proteins. Gray dots indicate proteins involved in other cellular functions. (D) Heat map of ZAP-70–associated ribosomal proteins from unstimulated or anti-IgM–activated primary CLL cells (adjusted P < .05). Red and blue indicate relatively high and low protein abundance, respectively. Each row analyzed depicts a technical replicate. (E) Heat map of ZAP-70–associated ribosomal proteins from unstimulated or anti-IgM–activated BJAB^ZAP-70 cells vs nonspecifically biotinylated proteins from BJAB^control cells (adjusted P < .05). Red and blue indicate relatively high and low protein abundance, respectively. Each row analyzed depicts a technical replicate. (F) Whole-cell lysates from primary CLL cells, unstimulated or stimulated with anti-IgM for 20 minutes, were immunoprecipitated (IP) with a ZAP-70 antibody or control IgG and immunoblotted (IB) with antibodies for ZAP-70, Syk, and RPS17. (G) Quantification of RPS17 bound to ZAP-70. The levels of RPS17 coimmunoprecipitated with ZAP-70 were normalized to total ZAP-70 levels. Anti-IgM–treated samples were then normalized to nontreated samples (n = 4). (H) Comparison of polysome profiles between unstimulated (blue) and anti-IgM–activated (20 minutes) (red) primary CLL samples. Vertical lines separating the curves indicate the fractions primarily extracted from 40s ribosome subunits, 60s to 80s ribosome subunits, and polysomes. (I) Proteins from sucrose gradient fractions in primary CLL cells were precipitated by 20% (weight-to-volume ratio) trichloroacetic acid and immunoblotted with ZAP-70, RPL29, and RPS17. Blue boxes contain unstimulated cells; red boxes contain anti-IgM–activated cells. Fractions 6 through 19 represent 40s subunits, 60s to 80s subunits, and polysomes, separated by vertical lines. (J) Quantification of relative ZAP-70 protein levels (relative to RPL17) in indicated fractions of primary CLL samples (n = 3). Anti-IgM–activated samples were normalized to unstimulated samples. (K) Graph showing the relative (to untreated NSC) fold change in OPP labeling (30 minutes) in unstimulated or anti-IgM–activated (20 minutes) primary CLL cells (n = 11) that were previously transfected with NSC siRNA or ZAP-70 siRNA. (L) Representative MYC and β-actin immunoblots of primary CLL cells monocultured for 24 hours after transfection with nonspecific siRNA or ZAP-70 siRNA. (M) Quantification of c-MYC levels in primary CLL cells proficient or depleted of ZAP-70. Graphs were generated using ImageJ (n = 4). (N) Representative immunoblots for MYC, ZAP-70, and β-actin of primary CLL cells monocultured for 24 hours after transfection with nonspecific siRNA or ZAP-70 siRNA. To induce BCR signaling, monocultured CLL cells were treated with bead-bound anti-IgM for 24 hours. *P < .05, **P < .01.