st two hybrid screening The entire coding sequence and deletion mutants of mouse COP1 were fused in frame to the GAL4 DNA binding domain of the pAS2 vector. The resulting bait plasmid was used to screen pACT human ref 1 K562 erythroleukemia libraries by the yeast two hybrid method in Y190 yeast cells. In vitro binding assay A cDNA fragment containing the C terminal domain of FIP200 was inserted into the pGEX vector in frame with Gluta thione S transferase. Expression and purification of GST fused proteins and the binding conditions were as described. Cell culture, transfection, retroviral infection, and treatment with UV NIH3T3 mouse fibroblasts, mouse embry onic fibroblasts, and 293T human embryonic kidney cells were cultured, transfected via the cal cium phosphate DNA precipitation method, and infected with retroviral vectors as described.
For treatment with UV, cells were washed with PBS twice, exposed to UV light in a UV Crosslinker, and incubated in a serum containing complete medium. In some cases, cells were treated with 5 uM MG132 before harvest. Plasmid construction The GFP fused protein Inhibitors,Modulators,Libraries expression vector, into which COP1 cDNAs were subcloned, was described previously. COP1 mutants were generated by PCR. Protein analyses Cell lysis, immunoprecipitation, sodium dodecyl sulfate polyacrylamide Inhibitors,Modulators,Libraries gel electrophoresis, and im munoblotting were performed as described. Two types of lysis buffer used in this study were EBC buffer containing 2000 KIU ml of aprotinin, 1mM PMSF, 0. 1 mM NaF, 0. 1 mM Na3VO4, and 10 mM B glycerophosphate, and SDS sample buffer.
In some cases, Inhibitors,Modulators,Libraries immunopreci pitated proteins were treated with phosphatase before im munoblotting. A rabbit polyclonal antibody to an HA epitope was obtained from Santa Cruz. A mouse monoclonal antibody to an HA epitope was purchased from Boehringer Mannheim. Rabbit polyclonal antibodies to ULK1 and Atg13 were from Sigma. Rabbit polyclonal anti bodies to LC3 and p62 were acquired from Medical Biological Laboratories. Rabbit polyclonal antibodies to FIP200, p53, and COP1 were generated using bacterially produced polypeptides in our laboratory. A rabbit polyclonal antibody to Atg101 was provided by Dr. Noboru Mizushima. Split GFP assay GFP was split into two domains, N terminal and C terminal. Each domain was fused to two molecules, and trans fected into cells as described above.
GFP signals were observed using phase contrast or fluorescence micros copy and measured with a flow cytometer. A human cDNA clone containing entire coding sequence of FIP200 was obtained from Kazasa Inhibitors,Modulators,Libraries DNA Research Insti tute. Tumorigenicity assay Cells were subcutaneously AV-951 injected into NOD SCID mice. After 3 weeks or 2. 5 months, mice were sacrificed and the size of the tumor was measured. Conclusion In this study, we found the interaction between FIP200 and though COP1. Ectopic expression of COP1 reduced one of the different forms of FIP200, suggesting that COP1 modulates FIP200 associated activities, which may con tribute to a variety o