Figure 3.
ZAP-70 constitutively binds to cytoskeleton proteins. (A) Venn diagram compares the ZAP-70-binding proteins identified from mass spectrometry (MS) in unactivated primary CLL cells and BJAB cell lines. Gene set enrichment analysis showing the gene ontology terms extracted from the unique 71 proteins (primary CLL cells MS and BJAB cell line MS, log2 fold-change (log2FC >1, adj P < .01). (B) The 71 unique proteins from panel A were plotted by the log2FC. x-axis, identified in primary CLL cells MS; y-axis, identified in BJAB cell line MS. Colors indicate the subgrouped functions of the proteins. Dots in gray are the proteins involved in other cellular functions. (C) Heat map of proteins presented in panel B (primary CLL cells MS and BJAB cell line MS, log2FC >2, adj P < .01). Purple and orange indicate relative high and low protein abundance, respectively. Each condition analyzed depicts 3 (primary CLL cells) or 2 (BJAB cell line) technical replicates. Proteins were ranked from top to bottom by average log2FC combine primary CLL cells MS and BJAB cell line MS. (D) Venn diagram comparing ZAP-70–binding proteins identified from MS in unactivated primary CLL cells using ZAP-70–specific antibody pull down, in unactivated BJAB cell lines using biotinylated–ZAP-70 streptavidin pull down and in unactivated Kasumi-2 cells using a ZAP-70 proximity dependent biotinylation strategy (Sadras et al12) (log2FC >2, adj P < .01). (E) Proposed model for tonic BCR signal regulating LCP1 serine(5) phosphorylation in UM-CLL. Created with BioRender.com. (F) Phospho-LCP1 (S5) and total LCP1 immunoblots of primary UM-CLL cells monocultured for 24 hours after nonspecific control siRNA or ZAP-70 siRNA transfection. For anti-IgM–stimulated samples, monocultured CLL cells were treated with bead-bound anti-IgM for 20 minutes. (G) Phosphorylated LCP1 levels (relative to total LCP1 levels) 24 hours after siRNA transfection in UM-CLL samples (n = 3). Statistical significance between samples was assessed by paired 2-tailed Student t tests. ∗∗P < 10−3. ns, not significant.

ZAP-70 constitutively binds to cytoskeleton proteins. (A) Venn diagram compares the ZAP-70-binding proteins identified from mass spectrometry (MS) in unactivated primary CLL cells and BJAB cell lines. Gene set enrichment analysis showing the gene ontology terms extracted from the unique 71 proteins (primary CLL cells MS and BJAB cell line MS, log2 fold-change (log2FC >1, adj P < .01). (B) The 71 unique proteins from panel A were plotted by the log2FC. x-axis, identified in primary CLL cells MS; y-axis, identified in BJAB cell line MS. Colors indicate the subgrouped functions of the proteins. Dots in gray are the proteins involved in other cellular functions. (C) Heat map of proteins presented in panel B (primary CLL cells MS and BJAB cell line MS, log2FC >2, adj P < .01). Purple and orange indicate relative high and low protein abundance, respectively. Each condition analyzed depicts 3 (primary CLL cells) or 2 (BJAB cell line) technical replicates. Proteins were ranked from top to bottom by average log2FC combine primary CLL cells MS and BJAB cell line MS. (D) Venn diagram comparing ZAP-70–binding proteins identified from MS in unactivated primary CLL cells using ZAP-70–specific antibody pull down, in unactivated BJAB cell lines using biotinylated–ZAP-70 streptavidin pull down and in unactivated Kasumi-2 cells using a ZAP-70 proximity dependent biotinylation strategy (Sadras et al12) (log2FC >2, adj P < .01). (E) Proposed model for tonic BCR signal regulating LCP1 serine(5) phosphorylation in UM-CLL. Created with BioRender.com. (F) Phospho-LCP1 (S5) and total LCP1 immunoblots of primary UM-CLL cells monocultured for 24 hours after nonspecific control siRNA or ZAP-70 siRNA transfection. For anti-IgM–stimulated samples, monocultured CLL cells were treated with bead-bound anti-IgM for 20 minutes. (G) Phosphorylated LCP1 levels (relative to total LCP1 levels) 24 hours after siRNA transfection in UM-CLL samples (n = 3). Statistical significance between samples was assessed by paired 2-tailed Student t tests. ∗∗P < 10−3. ns, not significant.

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