Activation of STAT5 in malignant WM B cells. (A) Baseline STAT phosphorylation was measured in freshly sorted CD19⁺CD138⁺ cells obtained from the bone marrows of patients with WM (n = 6) and MWCL-1, BCWM.1, and RPCI-WM1 cells. After fixation and permeabilization, tyrosine phosphorylation of STATs 1, 3, 4, 5, and 6 was determined via fluorescence-activated cell sorter analysis. Normalization for nonspecific antibody binding was performed by dividing the mean fluorescence intensity associated with the specific phospho-antibody signal by the mean fluorescence intensity (MFI) of the isotype control. Data are presented as the normalized MFI (ΔMFI). Significance of STAT5 phosphorylation relative to other STAT proteins was determined by the Student t test for both primary cells and WM cell lines. *P < .05; **P < .01. (B) Immunohistochemical staining of pSTAT5 (brown) in bone marrow sections obtained from 4 consenting patients with WM (WM1-WM4) and 4 normal bone marrows (NM1-NM4) was performed using a polyclonal anti-pSTAT5 antibody, as described in “Methods.” Slides were visualized on an Olympus Provus AX70 light microscope, and images shown are original magnification ×400. (C) Costaining of pSTAT5 (red) with CD20⁺-expressing (blue) and CD138⁺-expressing (yellow) B cells in WM bone marrow. (D) Immunoprecipitation for STAT5A (5A) and STAT5B (5B) was performed on unstimulated BCWM.1 (column B) and MWCL-1 (column M) cells followed by western blotting and detection of STAT5A, STAT5B, and pSTAT5.