The Co-relation Between ERα And P73α In Breast Cancer Cells

Objective:To investigate the physical and functional relation between ERα and p73α in thebreast cancer cells.Methods:MCF-7cells were cultured and passaged by using DMEM containing10%offetal calf serum. MCF-7cells were transfected with DNAs encoding ERα and p73α.1. The binding between ERα and p73α was tested by Co-IP: cells weretransfected with ERα and p73α plasmids for16-18hours. The proteasome inhibitorMG-132was added to a final concentration of20μM to prevent the degradation ofthe ERα protein or p73α protein and the cells were incubated with MG-132for anadditional6hours before harvesting. Cell lysates were completely mixed with ERαpolyclonal antibody to pull down ERα antigen, the ERα antigen-antibody complexwere combine with protein A agarose beads. Then protein A agarose beadsimmunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to PVDF membranes forimmunoblotting. Antibodies used in immunoblotting included anti-p73α monoclonalantibody to test the existence of p73α in the ERα complex.2. Co-effect between ERα and p73α in protein expression was examined by WB:cells were transfected with ERα and p73α plasmids and then harvested cell lysatesafter24hours, cell lysates were resolved by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) and transfer,block, then use anti-ERα polyclonalantibodies and anti-p73α polyclonal antibody to test their protein expression.Results:1. In the absence of MG-132(20μM), ERα and p73α binded to each other.2. When Co-transfecting of ERα and p73α DNA, ERα and p73α inhibited theirprotein expression each other.Conclusion:ERα and p73α could bind each other and could inhibite their protein expression each other in the breast cancer cells.