Figure 3.

YB-1 interacts with YB-1. (A) In vivo binding of YB-1 and Flag–YB-1. MCF-7 cells were transfected with plasmids expressing Flag–YB-1 or Flag vector. Expression of transfected plasmid was confirmed by immunoblotting with either anti-Flag antibody (left) or anti-YB-1 antibody (middle). The same extracts were immunoprecipitated (IP) with anti-Flag antibody; bound endogenous YB-1 was visualized by immunoblotting with anti YB-1 antibody (right). (B) In vitro binding of GST–YB-1 and HA–YB-1. The full-length form of HA–YB-1 was expressed in bacteria. Extracts from cells were used for pull-down experiments. Equal amounts of GST–YB-1 and translated forms were bound to glutathione–agarose and incubated with extracts including HA–YB-1. After binding, glutathione–Sepharose beads were washed and boiled with sample buffer. Bound proteins were separated by SDS–PAGE, blotted and visualized by staining with an anti-HA-antibody.