In vitro binding assays using GST fused FIP200 protein and cell lysate containing the ectopically sellekchem expressed HA tagged COP1 showed that COP1 and FIP200 interacted in vitro. Different forms of FIP200 protein were expressed in cultured mammalian cells To analyze the function of FIP200 in mammalian cells, we raised a rabbit polyclonal antibody to FIP200 using a polypeptide corresponding to the region isolated by the yeast two hybrid screening, which specifically reacted with endogenous FIP200 as well as ectopically expressed FIP200 protein by Wester blotting. In the lysate isolated from proliferating mammalian cells, our antibody recognized two forms of FIP200, the slower migrating form being more readily extracted from the cells.

Because we have previously showed that COP1 is involved in cellular re sponse mediated by UV stimulation, we examined whether UV might affect FIP200. Interestingly, UV stimulation altered the ratio between these two forms, proliferating cells contained the slower mi grating form more, while Inhibitors,Modulators,Libraries UV treatment decreased the expression of the slower migrating form and, instead, increased that of the faster migrating form. FIP200 is known to be modified by Inhibitors,Modulators,Libraries phosphorylation, which often affects mobility in SDS PAGE. To test this possibility, we extracted the protein from cells trea ted with UV and un treated cells in an SDS sample buf fer, isolated FIP200 by immunoprecipitation, and treated it with Brefeldin_A phosphatase in vitro. The result showed that the difference in mobility Inhibitors,Modulators,Libraries was not Inhibitors,Modulators,Libraries due to the level of phosphorylation although both forms were phosphorylated.

Currently, we do not know the exact molecular identity of these two variants, which might be generated by alternative splicing or other post translational modifications. FIP200 interacts selleck chemicals llc with COP1 in the cytoplasm of proliferating cells in response to UV stimulation We have so far not been successful in detecting the COP1 FIP200 complex in cell lysate by immunoprecipi tation immunoblotting. One possible explanation for this is that our antibody does not recognize the complex. Another possibility is that the COP1 FIP200 complex may not be efficiently eluted from the cells in a buffer suitable for immunoprecipitation. In fact, we identified different forms of FIP200 by Western blotting possibly due to alternative splicing and one of them was not efficiently extracted in a buffer for immunoprecipitation. To overcome these problems and to further investigate the interaction between COP1 and FIP200 in vivo, we performed a Split GFP analysis, in which GFP was split into two domains, N terminal and C terminal, and fused to two molecules, respectively. If these two molecules interact with each other in the cell, the GFP signal will be restored.