Serine pSTAT3 binds DNA. (A) Nuclear extracts from 7 CLL patients (CLL 2, 7, 10, and 19-22) were analyzed using EMSA, as described in “EMSA.” Binding of STAT3 to biotin-labeled DNA probes is shown (lanes 1, 3, 5, 7, 9, 11, and 13). To compete with the binding, an unlabeled STAT3 binding-site DNA probe was added to the reaction in 100 times molar excess (lanes 2, 4, 6, 8, 10, 12, and 14). The 2 STAT3-DNA complexes are marked by arrows. (B) Elimination of STAT3-DNA binding by antibodies to STAT3 or serine pSTAT3. Nuclear extract of CLL patient 9 was studied by EMSA. The assay was conducted with biotin-labeled STAT3 binding-site DNA probe (lane 1) or with unlabeled probe (lane 2), with the addition of mouse anti–human STAT3 antibodies (lane 3), mouse immunoglobulin G1 isotype control (lane 4), rabbit anti–human serine pSTAT3 (lane 5), or rabbit serum (lane 6). Vertical line indicates realignment of the gel's image. (C) Pull-down of STAT3 by biotin-labeled DNA probe. Nuclear extract from CLL 23 and 24 was incubated with biotin-labeled STAT3 binding-site DNA probe and agarose-conjugated streptavidin beads. The attached proteins were separated by SDS electrophoresis, and STAT3 and serine pSTAT3 were detected by Western blot analysis (Biotin-labeled probe). Incubation of nuclear extracts with unlabeled STAT3 binding-site DNA probes (Unlabeled probe) and agarose-conjugated streptavidin beads only (Beads) were used as negative controls. (D) ChIP assay of CLL cells. CLL cell-derived chromatin was immunoprecipitated with STAT3 or rabbit serum (control). The coimmunoprecipitated DNA was amplified by PCR using upstream promoter constructs of STAT3, waf1/p21, c-Myc, or the control gene RPL30. As shown, STAT3-regulated (STAT3, waf1/p21, and c-Myc), but not control (RPL30), genes were amplified. RPL30 as well as STAT3, waf1/p21, and c-Myc genes were detected in whole cell chromatin-extracted DNA (Input).