| IntroductionProtein kinase C ( PKC ) is a number of serine/ threonine kinase family which play central regulatory roles in a multitude of cellular processes, including cell proliferation, cell cycle progression, differentiation, apoptosis. Increasing evidence from studies using in vitro and in vivo systems suggests that PKC is a key regulator of critical cell cycle transitions, including cell cycle entry and exit and the Gl and G2 checkpoint. PKC - mediated control of these transitions can be negative or positive, depending on the timing of PKC activation during the cell cycle and on the specific PKC isozymes involved. PKC signaling has been implicated in the control of the G2/M transition mostly by the activated MPF (maturation promoting factor) Otherwise, PKC controls the G2/M transition by the cyclin - dependent kinase inhibitor (CKI). p21 is a key target of PKC -mediated cell cycle modulation. It may play a major role in G2/M transition via a mechanism involving blockade of cdk2/cyclinA activity, inhibition of cdc25 upregulation, accumulation of phosphorylation on cdc2, and suppression of cdc2/cyclin B activity. In this study, we showed that the protein levels of the cyclin - dependent kinase inhibitor p21 rapidly increased in the one - cell staged fertilized eggs treated with PMA. The cleavage from one - cell stage of the fertilized eggs into two - cell stage was inhibited.Protein kinase B is newly found as a serine/threonine protein kinase , It could have widely spread functions in cell cycle regulation. The activation of protein kinase B/AKT is thought to be a critical step in the phosphoinositide 3 -kinase pathway which regulates cell growth and differentiation. In this study,we showed that the PDK inhibitor LY294002 regulated the cellular localization of Materials1. KUNMING mice were supplied from the Department of Laboratory Animals, China Medical University2. Pregnant mare serum gonadotropin(PMSG) and human chorionic gona-dotropin ( hCG) were obtained from Tianjin Huafu Biological Products Research Institute and Shanghai Products Research Insititute, respectively.Anti - P21 mouse monoclonal antibody from Beijing Zhongshan BiotechnologyAnti - mouse or anti - rabbit IgG secondary antibody from Santa Cruz BiotechnologyLY294002 from Sigma Biotechnology TritonX -100 from BOEHRINGER MANNHEIN GmbH Fluorescein isothiocyanate ( FITC) conjugated anti - mouse IgG antibody was purchased from Beijing Zhongshan Biotechnology HEPES from E. Metck DarmstadtMethodsSuperovulation and collection of eggsFor superovulation, female KUNMING mice 4-5 week old were injected with pregnant mare serum gonadotropin ( PMSG) , and after 46 - 48 hours with human chorionic ginadotropin. ( hCG). One - cell fertilized eggs were collected on the next day from oviduct of females. Then one cell eggs were transferred to M16 for culture in incubator with an atmosphere of 5% CO2. According to the time point , collected G2 phase eggs .Western Blot AnalysisWe treated 200 fertilized eggs in a series of steps including lysing them in l0ul of lysis buffer(50mM Tris - Hcl pH 7. 5 , 250mM NaCl, 5mM EDTA, ImM DTT ,0. 1% Triton ,50mM sodium orthovanadate, lOOug/mg PMSF and TPCK, 50ug/ml TLCK, 1 ug/ml leupeptin pepstatin and aprotinin) , frozing them below - 70 , and then thawing them at room temperature for 10 min. We did the same steps for three times, so we could get the extraction using the stepsmentioned.Laemmli sample buffer was added to the extracted protein,which were then boiled for 5 min. The protein samples were separated by 12% SDS - PAGE and transferred to nitrocellulose membrane. The membranes were blocked in Tris -buffered saline (TBS; 20 mM Tris -HCl, 137 mM NaCl, pH 7.6) containing 5% non - fat dried milk (TBS/milk) for 30 min at 37 . Later the Membranes were incubated for 2 h at room temperature or overnight at 4 with p21 primary antibody in TBS/milk with 0. 1% Tween 20 , followed by washing the membrane in TBSt/milk for 5 min three times. Blots were then incubated for 1 h at...
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