This result is consis tent with the immunostaining result of polytene chromosomes which shows that CP190dBTB still associ ates with polytene chromosomes at many sites. selleck screening library The polytene staining results described above indicate that the CP190dBTB protein does not associate Inhibitors,Modulators,Libraries with the Su Mod 67. 2 complex at gypsy, which is sup ported by immunoprecipitation assays. We showed pre viously that proteins Inhibitors,Modulators,Libraries in the Su complex, such as Su and Mod 67. 2, co precipitated with Cp190. We precipitated the myc CP190dBTB protein with anti MYC from extracts of the y2 w ct6,P, CP1903 TM6B, Tb pupae and detected very weak sig nals of co precipitated Mod 67. 2, in contrast to precipitation of wildtype Cp190. The anti Myc and anti Cp190 immunoprecipitation reactions were specific since neither Cp190 nor Mod 67.
2 were precipitated from the y2 w ct6 pupae with anti Myc or with pre immune serum. The results indicate that association of the myc CP190dBTB with the Mod 67. 2 containing complex is significantly weaker than Entinostat wild type Cp190. Role of BTB domain in the association of Cp190 with multiple types of Cp190 containing insulator complexes Cp190 associates with diverse insulators including Su, CTCF and BEAF32. To more closely investigate the role of the BTB domain in association between Cp190 and the three types of Cp190 containing insulator complexes, we performed chromatin immuno precipitation assays. We tested Su associated gypsy loci, 1A2 and 62D, CTCF associated Fab 8, CTCF2, CTCF12, CTCF13, BXC100 and BXC114 loci, and BEAF32A or BEAF32B associated scs, BEAF A2, BEAF A3, BEAF AB3, BEAF B12, BEAF B13 and BEAF B16 loci.
We included a site in chromosome locus 1A6 as a negative control. Signals from all loci were normalized to the signal of Fab 8 to reveal the relative strength Inhibitors,Modulators,Libraries of association Inhibitors,Modulators,Libraries of Cp190 with tested sites in comparison with the association of Cp190 with the Fab 8 region. The results indicate that Cp190 associates with Su complexes at gypsy, 1A2 and 62D, but not with the 1A6 negative control region. Cp190 also associates with CTCF sites at Fab 8, CTCF12, BXC100, BXC114, but not at CTCF2 and CTCF13. Cp190 binds to BEAF32 sites at scs, A2, and B16, but not at A3, AB3, B12, and B13. Association with the tested regions is specific and we did not detect these sites in ChIP sam ples precipitated with pre immune serum. We next determined the binding of the myc CP190dBTB at the Cp190 positive sites and the negative control 1A6 site. The signal of myc CP190dBTB at Fab 8 is significantly higher than the Lapatinib 1A6 negative control region, suggesting that substantial amounts of the myc CP190dBTB protein lacking the BTB domain still associates with the Fab 8 region.