Figure 3.

Expression of s80^HER4 induces ligand-independent STAT5A phosphorylation. (A) HC11 cells serum-starved overnight, treated with PRL, HRG, or HB-EGF for 30 min. ErbB4 was immunoprecipitated with a polyclonal ErbB4 antibody; immunocomplexes were analyzed by Western blot using a monoclonal ErbB4 antibody. Molecular weights (kDa) are indicated at left. The predicted sizes of the membrane bound full-length ErbB4/HER4 and an 80-kDa cleavage product that is either membrane-bound or soluble (m80^HER4/s80^HER4) are indicated at right. (B) HC11 cells were stably transfected with pcDNA4 or pcDNA4-s80, encoding GFP-tagged s80^HER4. Pooled clones of stably transfected cells were grown in serum-free media overnight and then treated ± HB-EGF for 30 min. ErbB4 was immunoprecipitated using a polyclonal antibody. Immunoprecipitates were analyzed by Western with an antibody against phosphotyrosine. (C) Western analysis to detect tyrosine phosphorylation of ErbB1, ErbB2, or ErbB3 immunoprecipitates from HC11-GFP-s80 cells treated ± EGF or HRG where indicated. (D) Pools of HC11-pcDNA4 or HC11-GFP-s80 cells were serum-starved and then treated with PRL or HB-EGF for 30 min. Western analysis of ErbB4 or GFP immunoprecipitates to detect phosphotyrosine resides; Western analysis of STAT5A immunoprecipitates to detect total STAT5A and phosphotyrosine 694 STAT5A/B. (E) 3D culture of HC11 cells in growth factor-reduced Matrigel ± PRL. Cells were cultured 10 d, stained with DAPI, and photographed; DAPI and DIC images are shown. Arrows indicate lumen formation.