After the completion of tests, access for food and water was restored. The mice

Mutagenesis primer sequences are avail able upon request. pGEX4T Xic2 was generated by subcloning a PCR fragment of Xic2 using pCS2 −Xic2 as the template into the BamHI and SalI restriction sites of pGEX4T 1. Production of Xic2 antibody and other antibodies GST Xic2 expressed in BL21Star was purified ARN-509 using Glutathione Sepharose 4B beads according to the manufacturers instructions. Rabbit polyclonal antibody against GST Xic2 was gener ated by the University of Texas Health Science Center at San Antonio SACI antibody core facility. Anti PCNA mouse monoclonal antibody was purchased from Santa Cruz and anti GST XCyclin E antibody was a gift from Peter K. Jackson and Marc W. Kirschner. In vitro transcription and translation In vitro transcription and translation reactions were performed using the SP6 TNT coupled reticulocyte lysate system and 35S methionine from New England Nuclear.<br><br> Dephosphorylation and inhibition assays Dephosphorylation of protein samples was performed using lambda phosphatase, Reactions were incubated at 30 C for 30 min in the presence of 80 or 200 units of PPase, terminated by the addition of protein loading AT7519 価格 dye, and analyzed by SDS PAGE and phosphorimager. Roscovitine was used as previously described to inhibit CDK2 activity, Caffeine was used at a final concentration of 10 mM from fresh stocks of 75 mM in XB, GST pull down assays, immunoblotting, and immunoprecipiation GST pull down assays using egg extract and immuno blotting were performed as previously described except the binding reactions were conducted at 4 C for 2 hours, Rabbit serum against Xic2 was used to immuno precipitate and immunoblot Xic2 in extracts.<br><br> Anti cyclin E antibody was used to immunoprecipiate cyclin E from extracts while anti PCNA antibody was used for immu noblots. Normal rabbit serum was used as a control for immunoprecipitations. Degradation assay, nuclei spin down assay, and phosphorylation shift assay Degradation assays were performed as previously オーダー Alisertib des cribed with the following modifications. Proteins la beled with 35S methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng ul demembranated XSC or ΦX174 single stranded DNA, The reactions were ana lyzed by PhosphorImager and quantitation was performed using ImageQuant software, The percentage of protein remaining for each sample was de termined by normalizing the amount of protein at the 0 hr time point to 100%. Nuclei spin down assays were per formed as previously described with the following minor modifications. LSS was incubated with ubiquitin, 35S methionine labeled Xic2 at a final dilution of 1:15, and XSC for 90 min at 23 C. When indi cated, methyl ubiquitin was added to a final concentration of 3 ug ul.