Fig. 2.

Direct interaction between 4ICD and STAT5A is mediated by HER4 Y984. HEK 293T cells were transfected with the indicated expression vectors and cell lysates were prepared at 48 h. post-transfection in EBC buffer. For immunoprecipitations, 500 μg of cleared cell lysates were incubated with HER4 or STAT5A specific antibodies at 4 °C overnight. Immune complexes were recovered by adding Protein A sepharose to each immunoprecipitation reaction and incubating for 3 h at 4 °C, and finally eluted into 60 μl of NuPAGE LDS Sample Buffer with Reducing Agent. Twenty μl of eluted immunoprecipitation reactions or 20 μg of EBC lysate (Input) was probed by western blot using the indicated immunoblot (IB) antibodies.