Figure 5.

ER and c-ABL form a protein complex in vivo. (A) Transient transfection in 293T cells. Left panel: 293T cells were cotransfected with cDNA for ER-α and c-ABL. The cell lysates were then immunoprecipitated with an anti-c-ABL antibody or a control immunoglobulin class G. After gel separation, the coprecipitated ER and c-ABL were detected by using the corresponding antibodies. Right panel: The reciprocal approach after immunoprecipitation of ER. (B) Interaction of endogenous ER and c-ABL proteins in T47D cells by immunoprecipitation with anti-c-ABL (left panel) and anti-ER (right panel) antibodies, respectively. HC indicates heavy chain. (C) Colocalization of cellular ER (green) and c-ABL (red) in T47D cells by confocal immunofluorescence. The inset shows the amplified view of the area circled by the dotted square. Arrows point to examples of colocalization indicated by yellow fluorescence. The nuclei were stained blue with TOPRO 3. Bar, 10 µm. (D) The interaction between endogenous ER and c-ABL is enhanced by TAM. T47D cells were treated with TAM(Tam, 1 µM) or ethanol as the control vehicle (Ctrl) for 6 hours. Cell lysates were then subjected to immunoprecipitation with the anti-c-ABL antibody. Left panel: The cellular ER pulled down was detected by Western analysis. Right panel: Data were quantitated and normalized with respect to immunoprecipitated c-ABL.