The MAPK family of proteins is upregulated in DLBCL cell lines. (A) An unsupervised heat map comprising the MAPK pathway proteins exhibiting up- or downregulation in 9 DLBCL cell lines, when compared with normal B cells from reactive tonsils. The displayed matrix represents the log2 ratio of LC-MS/MS detected activity/expression of the kinases from each experimental DLBCL cell line relative to normal B lymphocytes, as depicted by the corresponding color scale. Each row represents a separate probe binding site for the indicated kinase, identified and quantified by LC-MS/MS, and each column represents a separate DLBCL cellular lysate. KiNativ data were clustered with Cluster software and visualized with Treeview.15 ABC-like DLBCLs are depicted in blue, and GCB-like DLBCLs are depicted in orange. Similar results were obtained in one additional KiNativ experiment. (B) Immunoblotting of GCK, p-MAP2K4, MAP2K4, p-JNK, and JNK total proteins in unmanipulated DLBCL cell lines. Similar results were obtained in 3 sets of independent blots. (C) Immunoblotting of GCK total protein in muscle cells, human germinal center (GC) B cells, tonsillar B cells, and unmanipulated DLBCL cell lines. Similar results were obtained in 3 sets of independent blots. (D) Normalized expression reflecting average densitometry readings of 3 independent experiments in which total GCK protein levels were quantified and normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) level for protein concentration. Quantification was performed using Image J software (NIH). (E) JNK kinase assay. JNK was immunoprecipitated from the indicated cell lines and tonsils and used to phosphorylate its target c-jun in the presence of ATP. p-c-jun is detected by immunobloting. (F) LC-MS/MS signal for JNK after pull-down with the KiNativ ATP-mimetic probe in B cells purified from human tonsils, and in DLBCL cell lines. KiNativ data (A) is representative of 2 independent experiments. Results in (B-C,E-F) are representative of 3 independent experiments. Results in (D) are the normalized average ± standard error of the mean of 3 independent experiments.
