Transfection of MF2000 with dominant-negative Stat3.

Transfection of MF2000 with dominant-negative Stat3 specifically inhibits Stat3 activity. MF tumor cells were stably transfected with HA-tagged Stat3 wild type (Stat3WT) or a dominant-negative Stat3 mutant (Stat3D). (A) Expression of exogenous Stat3. Transfectants were lysed and analyzed for HA expression by Western blotting. (B) Dominant-negative Stat3 inhibits DNA binding of Stat3. MF cells transfected with Stat3WT or Stat3D were stimulated with IFN-α for the indicated periods of time followed by lysis of the cells. Stat3 capable of binding to DNA was affinity purified by means of biotinylated hSIE oligos and identified by Western blotting with Stat3 antibody (top panel). Aliquots of the lysates were analyzed for tyrosine phosphorylation of Stat3 (middle panel) followed by reblotting of total Stat3 (lower panel). (C) Dominant-negative Stat3 does not affect Stat6 activation. MF cells transfected with Stat3WT or Stat3D were stimulated with IL-4, followed by lysis of the cells. Stat6 capable of binding to DNA was affinity purified by means of biotinylated IgE promotor oligos and identified by Western blotting with Stat6 antibody (top panel). Aliquots of the lysates were analyzed for tyrosine phosphorylation of Stat6 (middle panel) followed by reblotting of total Stat6 (lower panel). The experiments shown in panels B and C were repeated with WT6.2, WT6.12, D1, and D14 with similar results.