FIG. 4.
Interaction between RPA and hRIPβ. (A) hRIPα and hRIPβ interact with RPA in HEK293T cells. Cells were transiently transfected with a plasmid encoding FLAG-RPA in combination with GST, GST-hRIPα, or GST-hRIPβ. Whole-cell lysates (WCL) were precipitated with glutathione-Sepharose beads. The bead-bound proteins were eluted and immunoblotted (IB) with anti-FLAG antibody (upper panel) or anti-GST antibody (lower panel). (B) Interaction between FLAG-RPA p70 and endogenous (Endo.) RPA p32. Cells were transfected with either blank vector (lane 1) or a plasmid encoding FLAG-RPA p70 (lane 2). FLAG-RPA p70 was immunopurified (IP) with anti-FLAG antibody. To roughly compare the amount of bound RPA p32 protein with that of FLAG-RPA p70, the blotted membrane was simultaneously probed with anti-FLAG and anti-RPA p32 antibodies. IgG, immunoglobulin G. (C) Interaction of RPA with endogenous hRIPα and hRIPβ. HEK293T cells were immunoprecipitated with either anti-HA antibody (control) or anti-RPA p70 antibody, followed by immunoblotting with anti-hRIP antibody (upper panel), anti-RPA p32 antibody (middle panel), and anti-RPA p70 antibody (lower panel). (D) Identification of the regions of hRIPα required for RPA interaction. GST and GST-RPA protein were used for GST pull-down assays with 35S-labeled, in vitro-translated hRIP constructs. Bead-bound proteins were eluted and visualized by autoradiography. GST and GST-RPA proteins were visualized by Coomassie brilliant blue staining.