PMC

Pathways differentially phosphorylated between BL (black bars), FL (gray bars), and MCL (white bars). Kyoto Encyclopedia of Genes and Genomes pathways identified with proteins phosphorylated at a serine or threonine residue (A) and at a tyrosine residue (B). The significance of the identified pathway is represented as −log10(p) for each NHL subgroup.

Two-way hierarchical clustering of phosphorylated proteins. A: Hierarchical clustering of B-NHL cell lines on the basis of p-Ser/p-Thr proteins: the vertical black bar highlights differentially phosphorylated proteins in the GC-derived NHLs, whereas the vertical open bar highlights differentially phosphorylated proteins in the non–GC-derived NHLs. B: Hierarchical clustering of B-NHL cell lines on the basis of p-Tyr proteins: the vertical black bar highlights differentially phosphorylated proteins in the GC-derived NHLs. C: Evidence view of the STRING diagram obtained with proteins highlighted by the vertical black bar in B. Different line colors represent the types of evidence for the association: turquoise, databases; pink, experiments; black, co-experiments; and purple, homology.

Schematic representation of proteins involved in the BCR signaling pathways. A simplified network of proteins involved in the BCR signaling pathway. Several of the proteins (BTK, B-cell linker protein, PAG1, SYK, CD19, CD22, LCK, LYN, PLCγ, and caspase recruitment domain-containing protein 11) have been identified with p-Tyr either only in BL and FL cell lines or with a higher spectral count in BL and FL cell lines compared with MCL cell lines. The p-Tyr proteins are displayed with a small P symbol in a red circle. The design of this figure was aided by materials from ScienceSlides (VisiScience Inc, Chapel Hill, NC).

Overview of identification and quantification results of triplicate phosphoproteome enrichment. A: Distribution of the proteins identified with a p-Ser and/or p-Thr residue between the MOAC enrichments and pY-IP enrichments. Most of the p-Ser/p-Thr proteins were identified in MOAC enrichments. B: Distribution of the proteins identified with a p-Tyr residue between the MOAC enrichments and pY-IP enrichments. The sequential enrichment strategy improved considerably the identification of p-Tyr proteins. C: A total of 1701 phosphorylated proteins were identified only in MOAC enrichment, only in pY-IP, or in both types of enrichment. D: Of the identified phosphoproteins, 109 kinases were found only in MOAC enrichment, only in pY-IP, or in both types of enrichment. E: Venn diagram representing the repartition of proteins identified with p-Ser/p-Thr residues in the three different types of B-NHLs. Of 1667 p-Ser/p-Thr proteins, 960 (57.58%) were common to the three B-NHL types. F: Venn diagram representing the repartition of proteins identified with p-Tyr residues in the three different types of B-NHLs.

PAG1 knockdown results in increased BJAB proliferation and responsiveness to antigen stimulation. BJAB with scramble shRNA, black bars; BJAB with PAG1 shRNA, white bars. A: Western blot analysis control and its histogram of PAG1 knockdown in BJAB cell lines. B: The WST-1 assay demonstrates a significant increase of BJAB proliferation after 48 hours when PAG1 is stably knocked down. C: This result was confirmed by the increase of the number of colonies obtained after 14 days of culture in methylcellulose media. D: Antigenic stimulations of BJAB cells result in a significant increase of proliferation when PAG1 is stably knocked down. ^∗P < 0.05, ^∗∗P < 0.01. LPS, lipopolysaccharide.

PAG1 and LYN are overexpressed and phosphorylated in germinal center–derived cell lines. A: PAG1 expression in the 11 cell lines. B: Schematic representation of PAG1 tyrosine residue phosphorylation in different types of B-NHLs. The histogram widths represent the span of phosphopeptides containing a p-Tyr, whereas the histogram heights represent the abundance of the phosphorylation (spectral count). C: Western blot analysis of the immunoprecipitations done with the anti-PAG1 antibody (IP PAG1) or with the cocktail of the three anti-phosphotyrosine antibodies (IP p-Tyr). D: Western blot analysis of LYN expression and its phosphorylation at Y396 (induces the enzymatic activity) and at Y507 (inhibits the enzymatic activity) of cell lines representative of BL, FL, and MCL.