2.3. Immunoprecipitation and western blot analysis

Co-immunoprecipitation of STAT5A and 4ICD or 4ICD-Y984F was performed exactly as described previously [13], [17] using HEK 293T cells were transfected with pEFneo-STAT5A, p4ICD or p4ICD-Y984F using Lipofectamine with Plus Reagent (Invitrogen). At 2 days post-transfection, cell lysates were prepared in EBC buffer (50 mM Tris, pH 7.5, 50 mM Tris, pH 7.5, 0.5% NP-40, with 1 mM phenylmethylsulfonyl fluoride) and 500 μg of lysate was immunoprecipitated overnight at 4 °C with antibodies directed against HER4 (Santa Cruz; sc-283) or STAT5 (Santa Cruz; sc-835). Immune complexes were collected by incubating with Protein A sepharose (Roche) at 4 °C for 3 h. and eluted by boiling in 60 μl of NuPAGE LDS Sample Buffer (4X) (Life Technologies) containing NuPAGE Sample Reducing Agent (10X) (Life Technologies). Twenty μl of each immunoprecipitate was loaded in each lane of a NuPAGE 4–12% Bis–Tris Gel (Life Technologies). Total cell lysates were collected using RIPA Buffer (10 mM NaPO4, pH 7.2, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Na-deoxycholate, 1% Nonidet P40) containing Complete EDTA-free Protease Inhibitor Cocktail (Roche) and PhosSTOP phosphatase inhibitor (Roche) and western blot analysis was performed exactly as described previously [18] using the primary antibodies directed against HER4 (Abcam; E200), Stat5 (Santa Cruz; sc-835), and α-tubulin (Upstate Biotechnology; 05829).