Inhibition of STAT3 DNA-binding activity by cyclin K.

(A) Parental or cyclin K–expressing A375 cells were serum starved for 48 hours and stimulated for 30 minutes with OSM. Nuclear extracts were incubated with a biotinylated double-stranded Siem oligonucleotide corresponding to a specific STAT3 DNA-binding site. Samples were analyzed by Western blot probed with polyclonal antibodies directed against STAT3 phosphorylated on Tyr⁷⁰⁵(lanes 1-4). In parallel, aliquots of the nuclear extracts were subjected to Western blot analysis using anti-STAT3 phosphorylated Tyr⁷⁰⁵ (lanes 5-8, top) prior to being stripped and reprobed with anti-STAT3 antibodies (lanes 5-8, bottom). (B) Normal A375 cells were serum starved for 48 hours and stimulated for 30 minutes with OSM (10 ng/mL). A titration of purified GST protein (500 ng, lane 5), GST–cyclin K (lanes 1-3), or buffer alone (lane 4) was incubated for 30 minutes with 30 μg nuclear extract prior to the addition of biotin-labeled double-stranded oligonucleotide probe. Samples were analyzed for DNA-binding activity by Western blot using polyclonal STAT3 phospho-Tyr⁷⁰⁵ antibodies. (C) DNA binding of STAT3 present in A375 parental or cyclin K–expressing cells was determined by EMSA. Supershift of STAT3 was performed (lane 6) with the addition of 2 μg polyclonal STAT3 antibodies.