Stochiometry of the cyclin K–STAT3 complex.

(A) Control or cyclin K–expressing A375 cells were serum starved for 48 hours and stimulated with OSM (10 ng/mL) for the indicated times. Nuclear (lanes 1-6) or cytoplasmic (lanes 7-12) cell extracts were analyzed by Western blot analysis with polyclonal antibodies directed against STAT3 (middle panel), its Tyr⁷⁰⁵ phosphorylated forms (top panel), or cyclin K (bottom panel). (B) Purified GST–cyclin K and his-STAT3^716–770 proteins (5 μg) were allowed to interact in vitro as described above, and aliquots of the binding reactions were then fractionated through Superose-12 filtration as described in “Materials and methods.” Eluate fractions were collected, and the presence of GST–cyclin K and his-STAT3^716–770 was evaluated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting in each fraction. (C) A375 clones expressing cyclin K were stimulated for 15 minutes with OSM and nuclear extracts were prepared. Aliquots (300 μL) were subjected to Superose-12 filtration and STAT3 and cyclin K elutions were evaluated by SDS-PAGE and Western blotting in each fraction. Note that STAT3 (not cyclin K) was detected in some experiments in a complex with a size higher than 400 kDa but that this was not reproducible under the conditions used to detect the cyclin K association. (D) Following gel filtration of nuclear extracts, fractions corresponding to size 180 to 220 kDa (lanes 1, 3, 5, 7) and 110 to 140 kDa (lanes 2, 4, 6, 8) were analyzed by coimmunoprecipitation to detect the presence of a cyclin K–STAT3 complex. Fractions were immunoprecipitated with the indicated antibodies and analyzed by Western blot using antibodies directed against STAT3 proteins (lanes 1-4) or cyclin K (lanes 5-8).