Figure 1
RPA interacts with the C-terminus of HERC2. (a) The indicated HERC2 fragments with an N-terminal Myc- or FLAG-tag were used to examine RPA binding and intramolecular binding of HERC2. (b) HEK-293T cells were transfected with Myc-HERC2 fragments. Interaction between the fragments and endogenous HERC2 was assessed by immunoprecipitation (IP) with anti-Myc antibody followed by immunoblotting (IB) with the indicated antibodies. Inputs were also loaded. (c) HEK-293T cells were transfected with the indicated plasmids and the interaction between fragments F6 and F7 was assessed by immunoprecipitation and immunoblotting with anti-Myc and anti-FLAG antibodies as indicated. (d,e) HeLa-shHERC2 cells were co-transfected with the indicated HERC2 fragments and St2-RPA1 (d) or St2-RPA2 (e) together with other RPA subunits (pEGFP-RPA1 or HA-RPA2, and FLAG-RPA3), induced with Dox, treated with MG132, and subjected to immunoprecipitation with anti-Myc antibody or Strep-Tactin pulldown followed by immunoblotting with the indicated antibodies. The arrow head indicates non-specific band from IgG. (f) HeLa-shHERC2 cells were co-transfected with Myc-HERC2-F5 and St2-RPA2 as indicated, induced with Dox, treated or not with MG132, and subjected to immunoprecipitation followed by immunoblotting with the indicated antibodies.
