Electrophoretic mobility shift assay using a STAT binding probe to demonstrate activation of STAT5 in FDC-P1 cells in response to hIL-3. (A) Nuclear extracts were prepared from cells expressing IL-3Rα + hβc or IL-3RΔNT + hβc and stimulated with or without 1 nmol/L IL-3. Specificity of binding was confirmed by addition of 100-fold excess cold competitor oligonucleotide, or 100-fold excess of a mutant oligonucleotide, which cannot bind STAT protein. The binding protein was verified to be STAT5 and not STAT1 by addition of specific antibodies to each: only anti-STAT5 antibody gave a supershift. (B) Analysis of dose responses to IL-3 by IL-3Rα + hβc, IL-3RΔNT + hβc, IL-3RΔCT + hβc, hβc only, untransfected FDC-P1 cells (FD-), and untransfected FDC-P1 cells stimulated with mGM-CSF as a positive control (last lane).