Embryos were Y-27632 2HCL observed with an Olympus SZX12 stereomicroscope and photographed with an Olympus DP10 digital camera. In Vitro Transcription of Wee1 mRNA Wee1 cDNA in pBluescript was kindly provided by Dr. Monica Murakami. The control mRNA encoding exogenous luciferase protein was prepared Inhibitors,Modulators,Libraries as described previously. Experiments with kinase dead and wild type Wee1 mRNA were per formed with constructs provided by Dr. Paul Mueller. In the kinase dead mutant, the codon for lysine 239 was converted to the codon for arginine. Western Analysis Embryos were lysed in EB buffer. Samples were then resolved on SDS polyacrylamide gels transferred to a nitrocellulose membrane and blocked in 3% nonfat dry milk in TBS 0. 1%Tween or 5% BSA in TBS 0. 1% Tween.
Membranes were incubated in primary antibody against Cdc25A and cyclin E, and Wee1, diluted in 5% BSA TBS 0. 1% Tween overnight at 4 C. Membranes were washed then incubated in secondary antibody 1 10,000 in TBS 0. 1% Tween. Immunoreactive proteins were detected by chemilluminescence using an ECL Plus kit. Isolation and visualization Inhibitors,Modulators,Libraries of genomic DNA Embryos were injected with Wee1 or luciferase mRNA, collected at indicated times, and lysed in DNA digestion buffer. Lysates were incubated at 37 C for 2 hrs. Pro teinase K was then added to a final concentration of 100g/mL with continued incubation at 50 C for 4 hrs with slight intermittent manual agitation. Genomic DNA was phenol/chloroform extracted, and precipitated overnight at 20 C in 100% ethanol. Samples were resuspended in TE, and resolved on a 0.
7% agarose gel, and ethidium bro mide stained DNA was visualized and photographed under Inhibitors,Modulators,Libraries UV illumination. Assessment of nuclear morphology Embryos were collected at the times indicated, fixed in 4% paraformaldehyde, dehydrated through an ethanol series, cleared in CitriSolv, embedded in paraffin, sec tioned 7m thick, deparaffinized, rehydrated, and stained with 1g/ml DAPI. Serial sections were viewed and photographed on an Olympus AX70 fluorescence microscope equipped with a Color View 12 digital cam era. Assay for cleavage of PARP Embryos previously injected with Wee1 and luciferase mRNA or 34 Xic1 and p27Xic1CK proteins were col lected at desired stages, snap frozen on dry ice, and assayed for the cleavage of exogenous PARP. Specifically, embryos were homogenized in caspase extraction buffer.
Lysates were incubated with 2 ng/mL recombinant human PARP at 27 C for 15 min, resolved on an SDS polyacrylamide gel, and transferred to a nitrocellulose Inhibitors,Modulators,Libraries membrane. Anti PARP and HRP conjugated anti rabbit diluted Inhibitors,Modulators,Libraries in 10% nonfat dry milk in PBS were used as primary and secondary antibodies, respectively. Both full length and cleaved PARP proteins www.selleckchem.com/products/PF-2341066.html were detected by chemiluminescence using an ECL Plus kit. Immunoprecipitation Western analysis and kinase assays Embryos were injected with Wee1 or luciferase mRNA, collected at indicated times, and lysed in EB.