Dab2 modulates TGF-beta-stimulated growth regulation and signaling in primary mammary epithelial cells.Mammary epithelial cells were prepared from pregnant control (+/df) and dab2 conditional knockout (df/df) mice inheriting Sox2-Cre. The cells were treated with or without TGF-beta (10 ng/ml). (A) Growth of the cells was determined by WST assay over a 6-day period. Student-t test indicated that the difference in cell growth was statistically significant for at day 3 to 6 for dab2 (+/df) (p<0.005) but not dab2 (df/df) cells. (B) Protein lysates prepared from the primary cultures were analyzed by Western blots for Dab2, phospho-Smad2, E-cadherin, N-cadherin, phospho-Erk1/2, total Erk1/2, PCNA, Bcl-2, Bcl-xl, Bax, activated caspase-3, and beta-actin. (C) The relative protein level was quantified from the Western blots using NIH Image J software, and the values of the optical density (O.D.) critical markers (p-Smad2, p = Erk1/2, and Bcl-2) were compared. (D) Co-immunoprecipitation was performed to determine the association between Grb2 and Sos or Dab2. The primary cells were stimulated by TGF-beta for a time course of 0, 5, 15, 30, and 90 min. At each time point, the monolayer was washed with ice-cold PBS, lysed, and the post-nuclear supernatants were used for immunoprecipitation with antibodies against Grb2. The eluted proteins from the immunoprecipitation were separated by SDS-PAGE, and immunoprobed for Sos, Dab2, and Grb2. The immunoprecipitation experiments were performed twice and similar results were obtained. (E) Relative Dab2 and Sos protein amounts in the Western blot were estimated using NIH Image J program and the O.D. values were plotted.
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