Figure 2
pSTAT3Ser727 subcellular localization characterizes human primary CLL B cells. (a) Nuclear extracts from CLL-BC (#n upper panel, lower panel) or UT7 hematopoietic cell line were prepared and incubated with biotin-labeled STAT3-binding site DNA probes and streptavidin magnetic beads for pull-down assays. Attached proteins were separated by SDS-PAGE and STAT3 was detected by immunoblotting. NE and OP indicate nuclear extracts and oligo-pull-down extracts, respectively; 1:100 of total NE were loaded on each gel. NE were incubated with biotin-labeled SIEm67- (upper panel) or with SIEm67 (S), IRF (I)- or Oct (O)-DNA (lower panel) probes; lo and hi indicate low and high exposure of the immunoblot. UT7 hematopoietic cells were pre-incubated for 15 min in the absence or presence of phorbol myristyl acetate (P) as positive controls. α and β indicate STAT3α and STAT3β isoforms. (b–d) CLL-BC and N-BC were isolated, fixed, permeabilized and processed for fluorescence confocal microscopy as indicated. (b) Rabbit (rb) anti-pSTAT3Ser727 (pS3Ser727) antibody in the absence or presence of immunogen peptide (ipep), rabbit anti-total STAT3 (total S3) and isotype control rabbit immunoglobulins (rb Ig) were used in co-labelings with mitotracker, as indicated. DIC indicates differential interference contrast. (c) Mouse anti-pSTAT3Ser727 (pS3Ser727 mAb) and rabbit anti-mtND1 antibodies were used in co-labeling assays. Fluorescent probes were FITC-coupled anti-rabbit Ig (rb Ig-FITC, green), red-mitotracker or PE-coupled anti-mouse Ig (red), DAPI (blue). Line scan intensity profiles (bottom) of pS3Ser (red), mtND1 (green) and DAPI (blue) labeling along indicated axes (x1–y1; x2–y2) from gated areas. DAPI-positive nuclear (N) and DAPI-negative cytoplasmic (C) areas are schematized. Representative images from five patients (#n relates to Table I patient’s number) and five healthy donor samples are shown. Magnification, × 100. (d) Pearson’correlation coefficients were measured for the indicated fluorescent signals (rabbit anti-pSTAT3Ser727 versus mitotracker; mouse anti-pSTAT3Ser727 versus rabbit anti-mtND1; mouse anti-pSTAT3Ser727 versus DAPI). Twenty-five cells were randomly examined in confocal microscopy for each patient, and data from five and three patients were pooled, which corresponds to over 100 cells observed; **P<0.01)
