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Fig. 3. Blockade of Stat5 activation by cytochalasin D. Primary mammary epithelial cells were cultured on basement membrane and exposed to cytochalasin D for the indicated times. Prolactin was added to the cultures 15 minutes before preparing nuclear extracts for electrophoretic mobility shift assay (EMSA) (A,B) or detergent-soluble lysates for immunoprecipitation of prolactin signalling components (C). (A) Control cultures were either left untreated or were incubated with prolactin 15 minutes before harvesting. Extracts were subjected to EMSA with Stat5 oligodeoxynucleotides, in either the absence or the presence of 2 µg antibodies to Stat1, Stat3, Stat5 or control IgG. Notice that the mobility of the Stat5 band becomes almost completely supershifted in the presence of Stat5 antibodies. (B) Nuclear extracts of cells cultured with cytochalasin D or its carrier DMSO were assessed by EMSA for their ability to recognize Stat5 probe. Protein-DNA complexes were visualized by autoradiography (top) and quantified following storage phosphor image analysis (bottom). In the quantitative analysis, the levels of radioactivity in the samples of cytochalasin-treated cells are compared with those in extracts of cells treated with vehicle alone for the equivalent time and are plotted relative to the signal in control cultures treated with prolactin only. The data were obtained from three independent experiments. (C) Cell lysates were immunoprecipitated (IP) with antibodies to Stat5a or Stat5b. After separation by 6.25% SDS-PAGE, precipitated proteins were analysed by immunoblotting with antibodies for phosphotyrosine (4G10) or the appropriate precipitating antibody. The complete absence of Stat5 phosphorylation after 24 hours of cytochalasin-D treatment, in comparison to its residual DNA binding activity in (B), might reflect differences in the sensitivity of the assays.
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