Establishment of cell clones stably expressing S3DN and S3WT proteins. (A) SRB12-p9 cells were transfected with expression vectors as described in materials and methods. Whole cell extracts from cell clones (number shown above each lane) were subjected to Western blotting and probed sequentially with antibodies to the FLAG octapeptide, Stat3 (recognizing both α and β isoforms), and β-actin. (B) SRB12-p9, Neo, S3DN2 and S3WT6 cells (lane 1, 2, 3 and 4 respectively) were serum-starved for 2 days. Nuclear extracts were prepared and the EMSA assay was performed as described in Materials and Methods. In lane 5 nuclear extract from S3WT6 was pre-incubated with excess cold (unlabeled) Stat3 probe prior to the addition of labeled probe. Open arrow, expected location for Stat3α homodimer; closed arrow, unbound probe; dot, non-specific band. Results are representative of three separate experiments.