PRMT5 inhibition restores regulation to the BCR pathway. (A-B) PTPROt mRNA expression was measured by qRT-PCR in resting B cells (RB), immortalized B cells generated from 3 separate donors (D-9, D-22, D-27) (A) and at various time points after EBV infection of normal B cells from 2 separate donors (D-33 and D-25) (B). The bar graph shows normalized fold expression of PTPROt mRNA relative to normal B cells using GAPDH as internal control. (C) PTPROt expression assessed by confocal microscopy in resting B cells as well as in the fully immortalized lymphoblastoid cell line (D-9). (D) ChIP assay was performed on crosslinked chromatin from immortalized (D-5, D-22) and transformed B cells (C7M3) using anti-PRMT5 antibody, and the retained DNA was amplified by qPCR using PTPROt-specific primers and probe. Fold enrichment was calculated relative to the input sample. Error bars represent standard deviation of triplicate measurements. (E) PTPROt mRNA expression and protein levels were evaluated by qRT-PCR (top) and confocal microscopy (bottom) in 2 immortalized cell lines (D-5 and D-22) incubated for 24 hours with either DMSO control or a highly selective PRMT5 inhibitor, compound 5 (CMP5, 40 µM). (F) Phosphotyrosine proteins immunoprecipitated from whole-cell extracts of 60A cells incubated for 24 hours with either DMSO or CMP5 (40 µM) were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and probed with anti-SYK antibody (top left) and anti-pY(416)SRC and SRC antibodies (top right). Whole-cell extracts of 60A cells incubated for 24 hours with wither DMSO, CMP5 (40 µM), or ibrutinib (250 nM) were separated on SDS-PAGE and probed with anti-pBTK antibody, anti-BTK or GAPDH antibodies (bottom). *P < .05; **P < .01; ***P < .005. Error bars indicate SEM.