The resulting bait plasmid was used to screen pACT human 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. http://www.selleckchem.com/products/U0126.html 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 Inhibitors,Modulators,Libraries fibroblasts, mouse embry onic fibroblasts, and 293T human embryonic kidney cells were cultured, transfected via the cal cium Inhibitors,Modulators,Libraries 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. Anacetrapib Plasmid construction The GFP fused Inhibitors,Modulators,Libraries protein 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 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, 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 Inhibitors,Modulators,Libraries 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 DNA Research Insti tute. Tumorigenicity assay Cells were subcutaneously injected into NOD SCID mice. After 3 weeks or 2. 5 months, mice were selleck chemical Bicalutamide sacrificed and the size of the tumor was measured. Conclusion In this study, we found the interaction between FIP200 and COP1.