细胞信号转导的技术方法.ppt

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1、First Military Medical University细胞信号转导的技术方法细胞信号转导的技术方法First Military Medical UniversityFirst Military Medical University蛋白激酶磷酸化及其活性测定蛋白激酶磷酸化及其活性测定First Military Medical University蛋白激酶磷酸化的检测n n裂解培养的细胞获得裂解上清裂解培养的细胞获得裂解上清n n以免疫共沉淀法获得蛋白激酶以免疫共沉淀法获得蛋白激酶n nSDS-PAGEn n再加入蛋白激酶磷酸化特异性抗体再加入蛋白激酶磷酸化特异性抗体n n以以Pho

2、spho Ab-HRP Western 化学发光检化学发光检测试剂盒测定蛋白激酶的磷酸化程度。测试剂盒测定蛋白激酶的磷酸化程度。First Military Medical University经典蛋白激酶活性分析实验流程经典蛋白激酶活性分析实验流程n n以免疫共沉淀法获得蛋白激酶以免疫共沉淀法获得蛋白激酶n n加入该激酶底物和加入该激酶底物和 32-ATP，激酶使底，激酶使底物磷酸化物磷酸化n n放射自显影，确定磷酸化条带放射自显影，确定磷酸化条带First Military Medical Universityn n以免疫共沉淀法获得蛋白激酶以免疫共沉淀法获得蛋白激酶n n加入该激酶底物，

3、激酶使底物磷酸化加入该激酶底物，激酶使底物磷酸化n n再加入底物磷酸化特异性抗体再加入底物磷酸化特异性抗体n n以以Phospho Ab-HRP Western 化学发光化学发光检测试剂盒测定底物的磷酸化程度，进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非放射性蛋白激酶活性分析实验流程First Military Medical University较传统激酶活性测定方法的优点n n无需接触放射性物质无需接触放射性物质无需接触放射性物质无需接触放射性物质n n敏感度高：可以检测到每个磷酸化分子敏感度高：可以检测到每个磷酸化分子敏感度高

4、：可以检测到每个磷酸化分子敏感度高：可以检测到每个磷酸化分子n n特异性：利用磷酸化特异性抗体可以进行位点特异性：利用磷酸化特异性抗体可以进行位点特异性：利用磷酸化特异性抗体可以进行位点特异性：利用磷酸化特异性抗体可以进行位点特异性分析特异性分析特异性分析特异性分析n n信噪比高信噪比高信噪比高信噪比高n n低背景低背景低背景低背景n n系统完整：提供一整套直接从细胞裂解液中测系统完整：提供一整套直接从细胞裂解液中测系统完整：提供一整套直接从细胞裂解液中测系统完整：提供一整套直接从细胞裂解液中测试酶活的试剂试酶活的试剂试酶活的试剂试酶活的试剂n n节约时间：由几天降到几分钟节约时间：由几天降到

5、几分钟节约时间：由几天降到几分钟节约时间：由几天降到几分钟First Military Medical University024681012140 5 10 20 30 60 90 120 Time(min)Relative kinase activityDynamic processes of p38 activation by LPS in RAW cellsFirst Military Medical UniversityEffect of individual MAPK pathways on PRAK activity in intact cellsMKK7(D)GST-ATF2G

6、ST-ELK1MKK1(E)MKK6(E)ControlHSP27PRAKFirst Military Medical UniversityControlAniso.ArseniteTNF-80kDa-47kDa-39kDa-80kDa-47kDakDaControlAniso.ArseniteTNFABThe major kinases of p38 and p38First Military Medical UniversityP38 MAP Kinase PathwayFirst Military Medical University蛋白磷酸化位点分析及其蛋白磷酸化位点分析及其功能鉴定功

7、能鉴定First Military Medical UniversityPhosphopeptide map of HSP27 phosphorylated by PRAK in vitro ChromatographyElectrophoresis pH1.9+MAPKAPK2ChromatographyElectrophoresis pH1.9+PRAKChromatographyElectrophoresis pH1.9MIX+First Military Medical University+-+-HSP27p38 GST-PRAK(93A)GST-PRAK(WT)GST-PRAK(1

8、86A)GST-PRAK(212A 214A)GST-PRAK(182A)GST-PRAK(WT)GST-PRAK(182D)GST-PRAK(182D 212D)+-+-+-Fold of Activation05101520GST-PRAK(182A)GST-PRAK(182D)GST-PRAK(WT)GST-PRAK(93A)GST-PRAK(186A)GST-PRAK(182D 212D)GST-PRAK(212A 214A)BAElectrophoresis pH 8.9+p38-PRAK(182A)+p38-PRAK(182D)p38-PRAK(wt)T182 is the reg

9、ulatory phosphorylation site of PRAKFirst Military Medical UniversityDNA重组技术的应用重组技术的应用v活性诱变体v无活性诱变体v结构与功能的研究First Military Medical UniversityPCR primerPCR primerFirst Military Medical UniversityFirst Military Medical UniversityJNK2JNK3JNK1p38p38 p38 p38 ERK1ERK2ERK5The relationship between the membe

10、rs of MAPK familyFirst Military Medical UniversityMAPK Dural Phosphorylation Sites L-12 Length *hp38 DFGLARHTDD-EMTGYVATRWYRAPE25hP38 DFGLARQADE-EMTGYVATRWYRAPE25hp38 DFGLARQADS-EMTGYVVTRWYRAPE25hp38 DFGLARHADA-EMTGYVVTRWYRAPE25hJNK1 DFGLARTAGTS-FMMTPYVVTRYYRAPE27hJNK2 DFGLARTACTN-FMMTPYVVTRYYRAPE27

11、hJNK3 DFGLARTAGTS-FMMTPYVVTRYYRAPE27hERK1 DFGLARIADPEHDH-TGFLTEYVATRWYRAPE31hERK2 DFGLARVADPDHDH-TGFLTEYVATRWYRAPE31hBMK1 DFGMARGLCTSPAEH-QYFMTEYVATRWYRAPE32YHOG1 DFGLARIQDP-QMTGYVSTRWYRAPE 25YSMK1 DFGLARGIHAGFFKCHS-TVQPHITNYVATRWYRAPE36YMPK1 DFGLARGYSENPVEN-SQFLTEYVATRWYRAPE32YKSS1 DFGLARCLASSSDSRE

12、T-LVGFMTEYVATRWYRAPE35YFUS3 DFGLARIIDESAADNSEPTGQQSGMTEYVATRWYRAPE38domain VII VIIILoop-12(T-Loop)sequence of MAP kinasesFirst Military Medical UniversityConstruction of p38 loop-12 to ERK like structure p38 .DFGLARHTDDE-MTGYVATRWYRAPE.p38(E).DFGLARHTDDE-MTEYVATRWYRAPE.p38(6+).DFGLARHTDDEHDHTGFMTGYV

13、ATRWYRAPE.p38(6+E).DFGLARHTDDEHDHTGFMTEYVATRWYRAPE.p38(VAP).DFGLARVADPE-MTGYVATRWYRAPE.p38(DL).DFGLARHTDDD-LTGYVATRWYRAPE.p38(VAPD6+LE).DFGLARVADPDHDHTGFLTEYVATRWYRAPE.ERK2 .DFGLARVADPDHDHTGFLTEYVATRWYRAPE.First Military Medical UniversityMAPKMKKSubstratesLoop-12 is a key structure to determine the

14、selection of substrate (.TXY.)-First Military Medical University病毒技术对于揭示细胞信号病毒技术对于揭示细胞信号通路的作用通路的作用v腺病毒腺病毒v逆转录病毒逆转录病毒First Military Medical UniversityFirst Military Medical UniversityFirst Military Medical University细胞信号分子特异性抑制细胞信号分子特异性抑制剂的应用剂的应用First Military Medical University几种几种MAPKMAPK通路抑制剂的化学结构

15、示意图通路抑制剂的化学结构示意图:ONH2OCH3OPD98059HNNSOFCH3NOHNFNHNSB202190SB203580CH=CHCH=CHOHOCH3OHOCH3OOCurcuminFirst Military Medical UniversityEffects of SB203580 and PD98059 on endogenous PRAK activityHSP27TNF+-Arsenite+-PMA+-SB203580_ _-+-+-+PD98059-+-+-+-First Military Medical University细胞信号分子的荧光标记细胞信号分子的荧光标

16、记First Military Medical UniversityLPSEGFControlFirst Military Medical Universityp38p38 p38 p38 First Military Medical Universityp38p38 p38 p38 First Military Medical UniversityLPS Control UV First Military Medical UniversityFirst Military Medical University细胞信号分子的三维结构分细胞信号分子的三维结构分析析First Military Me

17、dical University蛋白质三维结构分析对于揭示信号分子的功能的作用First Military Medical UniversityFirst Military Medical University蛋白质与蛋白质相互作用的研究蛋白质与蛋白质相互作用的研究First Military Medical UniversityPHTHSH3SH2KinaseBtkSH3SH2KinaseSrc蛋白激酶结构域First Military Medical University1、蛋白质结合实验First Military Medical University2、免疫共沉淀实验First Mil

18、itary Medical University2、FRET 和 BRETFirst Military Medical University与信号分子相互作用蛋白质与信号分子相互作用蛋白质分子的相互作用分子的相互作用First Military Medical University酵母双杂合系统First Military Medical UniversityIdentification of MEF2C as a substrate for p38First Military Medical University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：PEC

19、AM：白细胞整合素：选择素：选择素配体：白细胞激活物：Ig家族成员图图12.1 白细胞的渗出过程白细胞的渗出过程 First Military Medical University噬菌体展示技术First Military Medical University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。First Military Medical UniversityFirst Military Medical University051015Relative luciferase activityControlLPSEGFLPS+FHPIp38 is involv

20、ed in the enhancement of TNF-promoter transactivityFirst Military Medical UniversityInduction of c-Jun through MEF2C phosphorylation by p38First Military Medical University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。First Military Medical UniversityEMSAFirst Military Medical University对细胞内信号分子的干预技术研究细胞内

21、蛋白质尤其是转录因子与特定核酸序列的相互作用。First Military Medical University反义核酸技术的应用First Military Medical UniversityRNAiFirst Military Medical UniversityRNAiFigure 1.Effects of mex-3 RNA interference on levels of the endogenous mRNA.Nomarski DIC micrographs show in situ hybridization of 4-cell stage embryos.(A)Negati

22、ve control showing lack of staining in the absence of the hybridization probe.(B)Embryo from uninjected parent showing normal pattern of endogenous mex-3 RNA(purple staining).(C)Embryo from parent injected with purified mex-3 antisense RNA.These embryos(and the parent animals)retain mex-3 mRNA,altho

23、ugh levels may be somewhat less than wild type.(D)Late 4-cell stage embryo from a parent injected with dsRNA corresponding to mex-3;no mex-3 RNA is detected.(Templates used for interfering RNA and in situ probes were largely non-overlapping.)First Military Medical University信号分子基因表达检测技术研究细胞内mRNA表达水平

24、。First Military Medical UniversityFirst Military Medical UniversityHeartBrainPlacentaLungLiverSkeletal MuscleKidneyPancreasp38p38 p38 p38 kb3.5-2.5-1.8-2.0-Tissue distribution of mRNA of p38 isoformsFirst Military Medical Universityp38、ATF2MEF2CCHOP10SAP1TNF-mRNA etc.monocytemacrophage Rt LPSMKK3/6MKKKsnucleusTK CD14PRAKsHSPCytoskeleton?LBP？First Military Medical UniversityTHANKSFOR YOUR ATTENTION