The mem branes were then probed with horseradish peroxidase conjugated anti mou

Alternatively, Xic2 phosphorylation by CDK2 may influ ence the binding of Xic2 to PCNA, DNA, or the ubiquitination machinery. The finding that CDK2 phos phorylation of Xic2 inhibits its PCNA dependent turn over suggests that Xic2 phosphorylation may be an important regulator of Xic2 stability and function. AP24534 価格 Conclusions In this study, we provide the first biochemical examin ation of the regulation of the Xenopus CDK inhibitors, p16Xic2 and p17Xic3, using the egg extract model system. Our studies indicate that Xic2 is targeted for DNA and PCNA dependent ubiquitination and degradation in the interphase egg extract and that this turnover of Xic2 is promoted by Cdt2 and inhibited by CDK2 dependent phosphorylation of Xic2 at residues Ser 98 and Ser 131.<br><br> Additionally, it appears that during conditions mimick ing a DNA damage AT7519 溶解度 checkpoint, Xic2 is targeted for phosphorylation by a caffeine sensitive kinase at residues Ser 78 and Ser 81, although the consequence of this phosphorylation is still unclear. Xic3 appears to be stable in the interphase egg extract in the presence or absence of DNA. In their initial discovery of Xic2, Daniels et al. de scribed Xic2 as an ortholog of mammalian p21 which is known to be transcriptionally induced by p53 upon DNA damage. p21 has also been shown to be a substrate of both CRL4Cdt2 and SCFSkp2, The RNA expres sion pattern of Xic2 in somites, the tail bud, the lens, and the cement gland suggest that Xic2 protein is expressed during late embryonic development, but how Xic2 protein may be regulated by proteolysis during development remains unknown.<br><br> It will be important to study how Xic2 may be regulated by buy Alisertib PCNA, CRL4Cdt2, or CDK2 during developmental patterning. It will also be necessary to study Xic1, Xic2, and Xic3 and their reg ulators in the context of the developing embryo and the somatic cell to fully understand how these three Xenopus CDK inhibitors mediate the events of early development and cell cycle control in the frog. Methods Preparation of Xenopus extracts and demembranated sperm chromatin Xenopus interphase extracts, stable 90 mitotic extract, and demembranated Xenopus sperm chromatin were prepared as pre viously described. All studies involving animals were conducted according to the rules established by the Universities of Federation for Animal Welfare, the World Society for the Protection of Animals Working Party, and the American Veterinary Medical Association.<br><br> This work was approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio which is accredited by the Association for Assessment and Ac creditation of Laboratory Animal Care under protocols 99045I 04 06 A and 11073x. Cell culture Xenopus Tissue Culture cells were propagated at room temperature in L 15 medium with L glutamine supplemented with 10% fetal bo vine serum and 50 ug ml of penicillin streptomycin, Cells were irradiated with 10 Gy using a 137Cesium source Mark I Model 68A irradia tor and harvested 4 and 8 hours later in RIPA buffer containing prote ase inhibitors, Generation of Xic2 mutants and other constructs All point mutants of Xic2 in pCS2 were generated by using pCS2 −Xic2 as the template and the QuickChange Site Directed Mutagenesis Kit followed by DNA sequencing to confirm the mutagenesis.