细胞信号转导的技术方法

FirstMilitaryMedicalUniversity细胞信号转导的技术方法FirstMilitaryMedicalUniversityFirstMilitaryMedicalUniversity蛋白激酶磷酸化及其活性测定FirstMilitaryMedicalUniversity蛋白激酶磷酸化的检测裂解培养的细胞获得裂解上清以免疫共沉淀法获得蛋白激酶SDS-PAGE再加入蛋白激酶磷酸化特异性抗体以PhosphoAb-HRPWestern化学发光检测试剂盒测定蛋白激酶的磷酸化程度。FirstMilitaryMedicalUniversity经典蛋白激酶活性分析实验流程以免疫共沉淀法获得蛋白激酶加入该激酶底物和32-ATP，激酶使底物磷酸化放射自显影，确定磷酸化条带FirstMilitaryMedicalUniversity以免疫共沉淀法获得蛋白激酶加入该激酶底物，激酶使底物磷酸化再加入底物磷酸化特异性抗体以PhosphoAb-HRPWestern化学发光检测试剂盒测定底物的磷酸化程度，进而推出蛋白激酶活性非放射性蛋白激酶活性分析实验流程FirstMilitaryMedicalUniversity较传统激酶活性测定方法的优点无需接触放射性物质敏感度高：可以检测到每个磷酸化分子特异性：利用磷酸化特异性抗体可以进行位点特异性分析信噪比高低背景系统完整：提供一整套直接从细胞裂解液中测试酶活的试剂节约时间：由几天降到几分钟FirstMilitaryMedicalUniversity02468101214051020306090120Time(min)RelativekinaseactivityDynamicprocessesofp38activationbyLPSinRAWcellsFirstMilitaryMedicalUniversityEffectofindividualMAPKpathwaysonPRAKactivityinintactcellsMKK7(D)GST-ATF2GST-ELK1MKK1(E)ControlHSP27PRAKFirstMilitaryMedicalUniversityControlAniso.ArseniteTNF-80kDa-47kDa-39kDa-80kDa-47kDakDa97.4-68.0-43.9-29.0-18.4-14.3-ControlAniso.ArseniteTNFABThemajorkinasesofp38andp38FirstMilitaryMedicalUniversityP38MAPKinasePathwayFirstMilitaryMedicalUniversity蛋白磷酸化位点分析及其功能鉴定FirstMilitaryMedicalUniversityPhosphopeptidemapofHSP27phosphorylatedbyPRAKinvitroChromatographyElectrophoresispH1.9+MAPKAPK2ChromatographyElectrophoresispH1.9+PRAKChromatographyElectrophoresispH1.9MIX+FirstMilitaryMedicalUniversity++++----+-HSP27p38+-+-+-FoldofActivation05101520BAElectrophoresispH8.9++p38-PRAK(182A)+p38-PRAK(182D)p38-PRAK(wt)T182istheregulatoryphosphorylationsiteofPRAKFirstMilitaryMedicalUniversityDNA重组技术的应用活性诱变体无活性诱变体结构与功能的研究FirstMilitaryMedicalUniversityPCRprimerPCRprimerFirstMilitaryMedicalUniversityFirstMilitaryMedicalUniversityJNK2JNK3JNK1p38p38p38p38ERK1ERK2ERK5TherelationshipbetweenthemembersofMAPKfamilyFirstMilitaryMedicalUniversityMAPKDuralPhosphorylationSitesL-12Length**hp38DFGLARHTDD-------------EMTGYVATRWYRAPE25hP38DFGLARQADE-------------EMTGYVATRWYRAPE25hp38DFGLARQADS-------------EMTGYVVTRWYRAPE25hp38DFGLARHADA-------------EMTGYVVTRWYRAPE25hJNK1DFGLARTAGTS-----------FMMTPYVVTRYYRAPE27hJNK2DFGLARTACTN-----------FMMTPYVVTRYYRAPE27hJNK3DFGLARTAGTS-----------FMMTPYVVTRYYRAPE27hERK1DFGLARIADPEHDH-------TGFLTEYVATRWYRAPE31hERK2DFGLARVADPDHDH-------TGFLTEYVATRWYRAPE31hBMK1DFGMARGLCTSPAEH------QYFMTEYVATRWYRAPE32YHOG1DFGLARIQDP-------------QMTGYVSTRWYRAPE25YSMK1DFGLARGIHAGFFKCHS--TVQPHITNYVATRWYRAPE36YMPK1DFGLARGYSENPVEN------SQFLTEYVATRWYRAPE32YKSS1DFGLARCLASSSDSRET---LVGFMTEYVATRWYRAPE35YFUS3DFGLARIIDESAADNSEPTGQQSGMTEYVATRWYRAPE38domainVIIVIIILoop-12(T-Loop)sequenceofMAPkinasesFirstMilitaryMedicalUniversityConstructionofp38loop-12toERKlikestructurep38...DFGLARHTDDE------MTGYVATRWYRAPE...p38(E)...DFGLARHTDDE------MTEYVATRWYRAPE...p38(6+)...DFGLARHTDDEHDHTGFMTGYVATRWYRAPE...p38(6+E)...DFGLARHTDDEHDHTGFMTEYVATRWYRAPE...p38(VAP)...DFGLARVADPE------MTGYVATRWYRAPE...p38(DL)...DFGLARHTDDD------LTGYVATRWYRAPE...p38(VAPD6+LE)...DFGLARVADPDHDHTGFLTEYVATRWYRAPE...ERK2...DFGLARVADPDHDHTGFLTEYVATRWYRAPE...FirstMilitaryMedicalUniversityMAPKMKKSubstratesLoop-12isakeystructuretodeterminetheselectionofsubstrate(...TXY...)---FirstMilitaryMedicalUniversity病毒技术对于揭示细胞信号通路的作用腺病毒逆转录病毒FirstMilitaryMedicalUniversityFirstMilitaryMedicalUniversityFirstMilitaryMedicalUniversity细胞信号分子特异性抑制剂的应用FirstMilitaryMedicalUniversity几种MAPK通路抑制剂的化学结构示意图:ONH2OCH3OPD98059HNNSOFCH3NOHNFNHNSB202190SB203580CH=CHCH=CHOHOCH3OHOCH3OOCurcuminFirstMilitaryMedicalUniversityEffectsofSB203580andPD98059onendogenousPRAKactivityHSP27TNF+++---------Arsenite+++---------PMA+++---------SB203580_---+--+---+PD98059----+--+--+-FirstMilitaryMedicalUniversity细胞信号分子的荧光标记FirstMilitaryMedicalUniversityLPSEGFControlFirstMilitaryMedicalUniversityp38p38p38p38FirstMilitaryMedicalUniversityp38p38p38p38FirstMilitaryMedicalUniversityLPS、UV刺激心肌细胞共聚焦显微镜大体扫描LPSControlUVFirstMilitaryMedicalUniversityFirstMilitaryMedicalUniversity细胞信号分子的三维结构分析FirstMilitaryMedicalUniversity蛋白质三维结构分析对于揭示信号分子的功能的作用FirstMilitaryMedicalUniversityFirstMilitaryMedicalUniversity蛋白质与蛋白质相互作用的研究FirstMilitaryMedicalUniversityPHTHSH3SH2KinaseBtkSH3SH2KinaseSrc蛋白激酶结构域FirstMilitaryMedicalUniversity1、蛋白质结合实验FirstMilitaryMedicalUniversity2、免疫共沉淀实验FirstMilitaryMedicalUniversity2、FRET和BRETFirstMilitaryMedicalUniversity与信号分子相互作用蛋白质分子的相互作用FirstMilitaryMedicalUniversity酵母双杂合系统FirstMilitaryMedicalUniversityIdentificationofMEF2Casasubstrateforp38FirstMilitaryMedicalUniversity第一步附着第二步激活第三步紧密粘附第四步渗出ECEC：趋化因子受体：PECAM：白细胞整合素：选择素：选择素配体：白细胞激活物：Ig家族成员图12.1白细胞的渗出过程FirstMilitaryMedicalUniversity噬菌体展示技术FirstMilitaryMedicalUniversity报告基因技术研究细胞信号转导通路通过转录因子对基因启动子转录活性的影响。FirstMilitaryMedicalUniversityFirstMilitaryMedicalUniversity051015RelativeluciferaseactivityControlLPSEGFLPS+FHPIp38isinvolvedintheenhancementofTNF-promotertransactivityFirstMilitaryMedicalUniversityInductionofc-JunthroughMEF2Cphosphorylationbyp38FirstMilitaryMedicalUniversity蛋白质与核酸相互作用技术研究细胞内蛋白质尤其是转录因子与特定核酸序列的相互作用。FirstMilitaryMedicalUniversityEMSAFirstMilitaryMedicalUniversi