FIG. 7.

DDB-1 physical interaction with CDT-1. (A) DDB-1 binding to CDT-1. FLAG-DDB-1, CDT-1-GFP, and LIN-23-MYC were expressed in 293T cells as noted by plus symbols (+) above the lanes. Numbers on top indicate the combinations of expression/coexpression. Anti-CDT-1 immunoprecipitation (IP), anti-FLAG IP, or anti-MYC IP were analyzed by Western blotting with anti-FLAG antibody (top panel), anti-CDT-1 antibody (bottom left panel), or anti-MYC antibody (bottom right panel). CDT-1-GFP expressed in 293T cells exhibited slow (120 kDa) and fast (100 kDa) migrating bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, similar to what is observed for ectopic expression of human Cdt1 in 293T cells (26). (B) SKPT-1-MYC binding to CDT-1 in 293T cells was performed similar to the method in panel A with the immunoprecipitations (IPs) performed with anti-CDT-1 or monoclonal anti-MYC antibodies. Rabbit polyclonal anti-MYC antibody was used to detect SKPT-1-MYC in the MYC immunoprecipitation (lanes 6 to 10, bottom panel) rather than the monoclonal anti-MYC antibody that is used in lanes 1 to 5, because SKPT-1-MYC runs at the same position as the mouse immunoglobulin G heavy chain. (C) DDB-1 and SKPT-1 binding to CDT-1 in vitro. ³⁵S-labeled FLAG-DDB-1 and SKPT-1-MYC were translated using a wheat germ extract system. DDB-1 was subsequently affinity purified using anti-FLAG antibody. On the left are autoradiographs showing in vitro translated ³⁵S-labeled FLAG-DDB-1 or SKPT-1-MYC bound by GST, GST-CDT-1, or CDK-phosphorylated GST-CDT-1 (GST-CDT-1P). Input lanes are 10% of the in vitro translated protein that was added to each GST binding reaction. On the right is a Coomassie-stained gel of the GST, GST-CDT-1, and phosphorylated GST-CDT proteins that were used in the binding assay. On the far right is a graph of the percentage of total input of ³⁵S-labeled FLAG-DDB-1 and MYC-SKPT-1 bound by GST-CDT-1 or GST-CDT-1P.