TOSO mRNA and protein expression detected by RT-PCR, immunoblotting, and flow cytometry. (A) TOSO mRNA expression in CD19⁺ peripheral B cells (n = 10), naive B peripheral B cells (n = 5), memory B cells (n = 3), and CD5⁺ B cells (n = 5) compared with purified CLL cells (n = 106). (B) TOSO is detected at 63 kDa due to posttranslational modification. Western blot using TOSO-specific 6B10 monoclonal antibody is shown for TOSO-pTRCHisTOPO recombinantly expressed in E coli, for patient CLL cells, and for TOSO-pGFP-C2 being transduced to HEK293 cells; cotransfection of TOSO-pGFP-C2 with TOSO-specific siRNA was performed for control of 6B10 antibody specificity. (C) TOSO protein expression is elevated in CLL cells compared with healthy donor CD19⁺ B cells; immunoblotting was performed using 6B10 monoclonal antibody; nontransformed TOP10 E coli served as negative control, and recombinantly expressed TOSO-pTRCHisTOPO served as positive control. Densitometry quotient of TOSO/tubulin is indicated accordingly. (D) Flow cytometry assessment of TOSO surface expression comparing healthy donor T and B cells to CLL patient T cells and malignant CLL B cells. (E) TOSO mRNA expression compared with other lymphoma entities: diffuse large B-cell lymphoma (DLBCL, n = 6), follicular lymphoma (FL, n = 6), marginal zone lymphoma (MZL, n = 2), T-cell non-Hodgkin lymphoma (T-NHL, n = 2), and Hodgkin lymphoma–derived cell lines (n = 4). Panel A, D, and E box plots show median, lst and 3rd quartiles, and whiskers (error bars) defined by, at most, 1.5× interquartile range (IQR).