Caspase 3 活性检测试剂盒

 产品编号: C1115
 产品包装: 20次
 产品价格: 496.00元
说明书下载

产品简介

产品简介:

产品编号 产品名称 产品包装 产品价格
C1115 Caspase 3 活性检测试剂盒 20次 496.00元

    Caspase 3 活性检测试剂盒(Caspase 3 Activity Assay Kit)是采用分光光度法检测细胞或组织裂
解液中Caspase 3酶活性或纯化的Caspase 3酶活性的试剂盒。
    Caspase(Cysteine-requiring Aspartate Protease)是一个在细胞凋亡过程中起重要作用的蛋白酶
家族。Caspase 3也称CPP32、Yama或apopain,有时被写作caspase-3或caspase3,属于caspase家族的CED-3亚家族(CED-3 subfamily),是细胞凋亡过程中的一个关键酶。Caspase 3是哺乳动物细胞中研究最多的一个caspase。Caspase 3可以剪切procaspase 3、6、7和9,并可以直接特异性剪切许多caspase底物,包括PARP(poly(ADP-ribose)polymerase),ICAD(Inhibitor of caspase-activated deoxyribonuclease),gelsolin和fodrin等。这些由caspase 3介导的蛋白剪切是细胞凋亡分子机制的重要组成部分。另外,caspase 3在细胞核凋亡过程中也起到了关键作用,包括染色质固缩(chromatin condensention),DNA片段化(DNA fragmentation)等。同时caspase 3对细胞起泡(Cell blebbing)也起到关键作用。
    本Caspase 3 活性检测试剂盒是基于casepase 3可以催化底物Ac-DEVD-pNA(acetyl-Asp-Glu-Val-
Asp p-nitroanilide)产生黄色的pNA(p-nitroaniline),从而可以通过测定吸光度来检测caspase 3的活性。pNA在405nm附近有强吸收。
    试剂盒中提供了caspase 3催化产生的黄色产物pNA,可以作为定量caspase 3酶活性的标准品。
    本试剂盒用酶标仪检测或容量不超过100μl的分光光度检测杯检测时,除标准曲线外可以检测20个
样品。
包装清单:

产品编号

产品名称

包装

C1115-1

裂解液

8ml

C1115-2

检测缓冲液

8ml

C1115-3

Ac-DEVD-pNA(2mM)

200μl

C1115-4

pNA(10mM)

200μl

说明书

1份

保存条件:
    -20℃保存,Ac-DEVD-pNA和pNA需避光保存。
注意事项:
    须自备可以测定A405或A400的酶标仪或容量不超过100μl的分光光度检测杯及相应分光光度计。优
先考虑测定A405,如有困难可以测定A400。
    Ac-YVAD-pNA需尽量避免反复冻融,请注意适当分装。
    测定蛋白浓度需Bradford蛋白浓度测定试剂盒(P0006),可向碧云天订购。建议样品用水稀释1倍后
再用Bradford法测定蛋白浓度,以降低DTT对蛋白浓度测定的干扰。
    pNA(中文名为4-硝基苯胺)有毒,请注意小心防护。pNA(10mM)在4℃、冰浴等较低温度情况下会凝
固而粘在离心管管底、管壁或管盖内,可以20-25℃水浴温育片刻至全部融解后使用。
    本试剂盒的裂解液可以和碧云天生产的其它Caspase活性检测试剂盒的裂解液通用,即本试剂盒裂
解液制备的蛋白样品可以用于碧云天其它Caspase活性检测试剂盒的检测。
    为了您的安全和健康,请穿实验服并戴一次性手套操作。

使用说明

使用说明:
1.准备工作:
  A.裂解液溶解后混匀并置于冰浴上备用。
  B.检测缓冲液溶解后混匀并置于冰浴上备用。
2.测定pNA标准曲线:
  A.标准品稀释液的配制:按照每0.9ml检测缓冲液加入0.1ml裂解液的比例配制适量的标准品

  稀释液。
  B.把试剂盒提供的pNA (10mM)用标准品稀释液稀释为0、10、20、50、100和200μM,作为

  标准品。
  C.每个浓度取100μl用酶标仪进行检测,或取适当量用容量不超过100μl的分光光度检测杯

  进行检测,测定A405。
  D.每一个标准品的A405减去不含pNA的空白对照的A405计算出实际的因pNA而导致的吸光度,

  并制作出 pNA浓度相当于A405的标准曲线。
3.样品的收集:
  A.对于悬浮细胞:把没有诱导凋亡的对照样品和诱导凋亡的样品,600g 4℃离心5分钟收

  集细胞,小心吸除上清,同时确保尽量没有细胞被吸除,PBS洗涤一次。同前吸尽上清后,

  按照每200万细胞加入100微升裂解液的比例加入裂解液,重悬沉淀,冰浴裂解15分钟。

  下转步骤3D。
  B.对于贴壁细胞:吸取细胞培养液,备用。用胰酶消化贴壁细胞,并收集至备用的细胞培

  养液中。600g 4℃离心5分钟收集细胞,小心吸除上清,同时确保尽量没有细胞被吸除,

  PBS洗涤一次。同前吸尽上清后,按照每200万细胞加入100微升裂解液的比例加入裂解液,

  重悬沉淀,冰浴裂解15分钟。下转步骤3D。
  C.对于组织样品:按照每3-10mg组织加入100微升裂解液的比例加入裂解液,在冰浴上用玻

  璃匀浆器匀浆。然后把匀浆液转移到1.5ml离心管中,冰浴再裂解5分钟。
  D.4℃ 16,000-20,000g离心10-15分钟。
  E.把上清转移到冰浴预冷的离心管中。
  F.立即测定caspase 3的酶活性或-70℃保存样品。同时可以取少量样品用Bradford法测定

  蛋白浓度,尽量使蛋白浓度达到1-3mg/ml,相当于每10微升待测样品中含有10-30μg蛋白。

  如果细胞较小,可以适当增加细胞的用量。
4.caspase 3酶活性的检测:
  A.取出pNA和适量的Ac-DEVD-pNA(2mM),置于冰浴上备用。
  B.如下设置反应体系:

 

空白对照

样品

检测缓冲液

90μl

80μl

待测样品

0μl

10μl

Ac-DEVD-pNA(2mM)

10μl

10μl

总体积

100μl

100μl

    注意:在设置反应体系时先加检测缓冲液,再加待测样品,适当混匀,注意避免在混匀

  时产生气泡。随后再加入10μl Ac-DEVD-pNA(2mM)。
  C.加入Ac-DEVD-pNA(2mM)后混匀,注意避免在混匀时产生气泡。37℃孵育60-120分钟。发现

  颜色变化比较明显时即可测定A405。如果颜色变化不明显,可以适当延长孵育时间,甚至

  可以孵育过夜。
  D.样品的A405扣除空白对照的A405,即为样品中caspase 3催化产生的pNA产生的吸光度。通过

  同步骤1中获得的标准曲线的对比就可以计算出样品中催化产生了多少量的pNA。
  E.参考Chemicon公司的caspase 3酶活力单位的定义:One unit is the amount of enzyme that

   will cleave 1.0 nmol of the colorimetric substrate Ac-DEVD-pNA per hour at 37℃

  under saturated substrate concentrations。即一个酶活力单位定义为当底物饱和时,

  在37℃一个小时内可以剪切1nmol Ac-DEVD-pNA产生1nmol pNA的caspase 3的酶量。这样就可以计

  算出样品中含有多少个酶活力单位的caspase 3。说明:在本试剂盒的检测体系中,底物的起始

  浓度为0.2mM,此时底物是饱和的,对于许多样品而言在37℃孵育2个小时以内底物都是饱和

  的;对于样品中caspase 3酶活力特别高的情况,须用裂解液适当稀释样品后再进行测定。
  F.用Bradford法检测待测样品中的蛋白浓度(由于裂解液中含有较高浓度的DTT,不适合采用

  BCA法进行蛋白浓度测定)。这样就可以计算出一个样品单位重量蛋白中所含的caspase 3的

  酶活力单位。

常见问题:
1.测定出的A405过低:
  A.样品中蛋白含量太低,裂解样品时需设法使样品中的蛋白浓度接近1-3mg/ml。
  B.样品中激活的caspase水平很低。首先确认凋亡现象是否明显,如果凋亡比较明显并且确

  认该caspase是可以被激活的,可以适当调节诱导细胞凋亡的时间,希望能找到一个caspase

  激活比较强的时间点,这样就可以检测出该caspase的激活。可以作一时间曲线,例如诱导

  凋亡0、2、4、8、16和24小时,或0、1、2、4、8和16小时,或0、1、2、4、6和8小时等。

  具体的诱导凋亡时间需根据具体情况而定。
  C.通过上述优化后A405还是比较低,或浓缩样品或做时间曲线比较困难,可以在测定样品时

  加大样品的用量,最多可达40μl。
    假设每次测定样品的用量为xμl,则:
    C1.此时标准品稀释液需按如下方法配制:按照xμl裂解液加入检测缓冲液至最终体积

  为100μl的比例配制适量的标准品稀释液。例如样品用量为40μl,则按照40μl裂解液加

  入60μl检测缓冲液,即0.4ml裂解液加入0.6ml检测缓冲液的比例配制标准品稀释液。其余

  标准曲线的测定方法同上面的使用说明所述。
    C2.此时如下设置反应体系:

 

空白对照

样品

检测缓冲液

90μl

(90-x)μl

待测样品

0μl

xμl

Ac-DEVD-pNA(2mM)

10μl

10μl

总体积

100μl

100μl

    说明:其中x不超过40,其余检测方法同上面的使用说明所述。

 

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51.Shi X, Gu A, Ji G, Li Y, Di J, Jin J, Hu F, Long Y, Xia Y, Lu C, Song L, Wang S, Wang X
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52.Chen X, Ran ZH, Tong JL, Nie F, Zhu MM, Xu XT, Xiao SD.
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53.Huang B, Cao K, Li X, Guo S, Mao X, Wang Z, Zhuang J, Pan J, Mo C, Chen J, Qiu S.
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54.Tan C, Zhang LY, Chen H, Xiao L, Liu XP, Zhang JX.
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55.Du K, Zheng Q, Zhou M, Zhu L, Ai B, Zhou L
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56.Xu Y, Zheng H, Kang JS, Zhang L, Su J, Li HY, Sun LK
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57.Feng Q, Cao HL, Xu W, Li XR, Ren YQ, Du LF
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58.Lou H, Fan P, Perez RG, Lou H.
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59.Zhang W, Liu J, Sun R, Zhao L, Du J, Ruan C, Dai K.
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60.Chen H, Mei L, Zhou L, Shen X, Guo C, Zheng Y, Zhu H, Zhu Y, Huang L.
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61.Yu G, Li Y, Tian Q, Liu R, Wang Q, Wang JZ, Wang X.
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62.Tian LL, Yue W, Zhu F, Li S, Li W.
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63.Xu B, Xu Z, Xia T, He P, Gao P, He W, Zhang M, Guo L, Niu Q, Wang A.
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64.Li M, Ding Y, Mu Y, Ao J, Chen X
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65.Wang K, Cheng L, Liang Y, Liu D, Li K, Wang P.
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66.Wu Y, Xia ZY, Dou J, Zhang L, Xu JJ, Zhao B, Lei S, Liu HM
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67.Tong JS, Zhang QH, Huang X, Fu XQ, Qi ST, Wang YP, Hou Y, Sheng J, Sun QY.
   Icaritin causes sustained ERK1/2 activation and induces apoptosis in human endometrial
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68.Li J, Cheng Y, Qu W, Sun Y, Wang Z, Wang H, Tian B
   Fisetin, a dietary flavonoid, induces cell cycle arrest and apoptosis through activation
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   Basic Clin Pharmacol Toxicol. 2011 Feb;108(2):84-93
69.Gong L, Liu FQ, Wang J, Wang XP, Hou XG, Sun Y, Qin WD, Wei SJ, Zhang Y, Chen L,
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70.Li P, Yin Y, Yu Q, Yang Q.
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71.Abdou MA, He Q, Wen D, Zyaan O, Wang J, Xu J, Baumann AA, Joseph J, Wilson TG, Li S,
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72.Li ZL, Liu JC, Hu J, Li XQ, Wang SW, Yi DH, Zhao MG
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73.Nie X, Song S, Zhang L, Qiu Z, Shi S, Liu Y, Yao L, Zhu D.
   15-Hydroxyeicosatetraenoic acid (15-HETE) protects pulmonary artery smooth muscle cells
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   Prostaglandins Other Lipid Mediat. 2012 Jan;97(1-2):50-9.
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74.Arockiaraj J, Easwvaran S, Vanaraja P, Singh A, Othman RY, Bhassu S.
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   caspase3c expression and activity in freshwater prawn Macrobrachium rosenbergii.

   Fish Shellfish Immunol. 2012 Jan;32(1):161-9. doi: 10.1016/j.fsi.2011.11.006.
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75.Zhao D, Lin F, Wu X, Zhao Q, Zhao B, Lin P, Zhang Y, Yu X
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   Toxicol In Vitro. 2012 Jun;26(4):595-602. doi: 10.1016/j.tiv.2012.02.004.
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76.Zhang W, Zhao L, Liu J, Du J, Wang Z, Ruan C, Dai K.
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77.Lv M, Zhang Y, Liang L, Wei M, Hu W, Li X, Huang Q.
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78.Lv L, Jiang SS, Xu J, Gong JB, Cheng Y
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79.Jia Y, Li Y, Du S, Huang K.
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80.Ma J, Zhang L, Han W, Shen T, Ma C, Liu Y, Nie X, Liu M, Ran Y, Zhu D.
   Activation of JNK/c-Jun is required for the proliferation, survival, and angiogenesis
   induced by EETin pulmonary artery endothelial cells.

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81.Wang YW, Wang SJ, Zhou YN, Pan SH, Sun B.
   Escin augments the efficacy of gemcitabine through down-regulation of nuclear factor-κB
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82.Tang J, Guo YS, Zhang Y, Yu XL, Li L, Huang W, Li Y, Chen B, Jiang JL, Chen ZN.
   CD147 induces UPR to inhibit apoptosis and chemosensitivity by increasing the
   transcription ofBip in hepatocellular carcinoma.

   Cell Death Differ.2012 Nov;19(11):1779-90.doi:10.1038/cdd.2012.60.Epub 2012 May 18.
83.Jiang Y, Lv H, Liao M, Xu X, Huang S, Tan H, Peng T, Zhang Y, Li H.
   GRP78 counteracts cell death and protein aggregation caused by mutant huntingtin
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84.Ye Z, Guo Q, Xia P, Wang N, Wang E, Yuan Y.
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   Brain Res.2012 Jun 29;1463:63-74.doi:10.1016/j.brainres.2012.04.050.Epub 2012 May 3.
85.Liao XH, Chen GT, Li Y, Zhang L, Liu Q, Sun H, Guo H.
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86.Chi J, Zhu Y, Fu Y, Liu Y, Zhang X, Han L, Yin X, Zhao D.
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   Mol Cell Biochem. 2012 Aug;367(1-2):227-36. doi: 10.1007/s11010-012-1336-5.
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87.Zhang S, Wan Y, Pan T, Gu X, Qian C, Sun G, Sun L, Xiang Y, Wang Z, Shi L
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88.Liu Q, Xiao L, Yuan D, Shi X, Li P.
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89.Zhu S, Wang Y, Jin J, Guan C, Li M, Xi C, Ouyang Z, Chen M, Qiu Y, Huang M, Huang Z
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90.Gao Q, Qi L, Wu T, Wang J.
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91.Ren Y, Cai Y, Jia D.
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   Cell Biochem Biophys. 2012 Nov;64(2):137-45. doi: 10.1007/s12013-012-9382-x.
92.Zhang L, Ma J, Shen T, Wang S, Ma C, Liu Y, Ran Y, Wang L, Liu L, Zhu D.
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   Cell Signal.2012 Oct;24(10):1931-9. doi: 10.1016/j.cellsig.2012.06.007.
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93.Xu HL, Yu XF, Qu SC, Qu XR, Jiang YF, Sui da Y
   Juglone, from Juglans mandshruica Maxim, inhibits growth and induces apoptosis in
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94.Qian Y, Guan T, Huang M, Cao L, Li Y, Cheng H, Jin H, Yu D.
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95.Zhu X, Li Y, Luo X, Fei J
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   Cell Signal. 2012 Jun;24(6):1134-40. doi: 10.1016/j.cellsig.2012.01.016.
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96.Hu W, Chen FH, Yuan FL, Zhang TY, Wu FR, Rong C, Jiang S, Tang J, Zhang CC, Lin MY.
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   Inflamm Res.2012 Apr;61(4):327-35.doi:10.1007/s00011-011-0414-6.Epub 2012 Jan 12.
97.Zhou Z, Wan Y, Zhang Y, Wang Z, Jia R, Fan Y, Nie H, Ying S, Huang P, Wang F
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   proliferator-activated receptor γ coactivator-1 alpha in ovaries of fetal and neonatal
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98.Hu R, Zhou P, Peng YB, Xu X, Ma J, Liu Q, Zhang L, Wen XD, Qi LW, Gao N, Li P.
   6-Shogaol Induces Apoptosis in Human Hepatocellular Carcinoma Cells and Exhibits
   Anti-TumorActivity In Vivo through Endoplasmic Reticulum Stress.

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99.Li Y, Liu L, Niu Y, Feng J, Sun Y, Kong X, Chen Y, Chen X, Gan H, Cao S, Mei Q.
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   Eur J Nutr.2012 Feb;51(1):107-17.doi:10.1007/s00394-011-0194-3.Epub 2011 Apr 24.
100.Dan Zhu D, HeHJ, Hao LR, TangJ, CaoD
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101.Gao W, Ji L, Li L, Cui G, Xu K, Li P, Tang B.
   Bifunctional combined Au-Fe(2)O(3) nanoparticles for induction of cancer cell-specific
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102.Cheng J, Tang W, Su Z, Guo J, Tong L, Wei Q.
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103.Zhai FX, Liu XF, Fan RF, Long ZJ, Fang ZG, Lu Y, Zheng YJ, Lin DJ.
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104.Hu M, Xu L, Yin L, Qi Y, Li H, Xu Y, Han X, Peng J, Wan X.
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105.Zhou YC, Liu B, Li YJ, Jing LL, Wen G, Tang J, Xu X, Lv ZP, Sun XG.
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106.Li Y, Wang H, Yang B, Yang J, Ruan X, Yang Y, Wakeland EK, Li Q, Fang X.
   Influence of carbon monoxide on growth and apoptosis of human umbilical artery smooth
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107.Guo C, Zeng X, Song J, Zhang M, Wang H, Xu X, Du F, Chen B.
   A Soluble Receptor for Advanced Glycation End-Products Inhibits Hypoxia/Reoxygenation-
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108.Zhang WF, Xu YY, Xu KP, Wu WH, Tan GS, Li YJ, Hu CP.
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110.Guo E, He Q, Liu S, Tian L, Sheng Z, Peng Q, Guan J, Shi M, Li K, Gilbert LI, Wang J,
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111.Xiao F, Zhang JS, Zhao JY, Wu D.
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112.Mao Z, Xia W, Wang J, Chen T, Zeng Q, Xu B, Li W, Chen X, Xu S.
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114.Yu W, Wu J, Cai F, Xiang J, Zha W, Fan D, Guo S, Ming Z, Liu C.
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115.Wang J, Wang Q, Li J, Shen Q, Wang F, Wang L.
   Cadmium induces hydrogen peroxide production and initiates hydrogen peroxide-dependent
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   Comp Biochem Physiol C Toxicol Pharmacol.2012 Nov;156(3-4):195-201.
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117.Cao M, Tong Y, Lv Q, Chen X, Long Y, Jiang L, Wan J, Zhang Y, Zhang F, Tong N.
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   Endoplasmic Reticulum Stressthrough Enhanced Fatty Acid Oxidation.

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118.Liu Q, Peng YB, Qi LW, Cheng XL, Xu XJ, Liu LL, Liu EH, Li P.
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119.Yue R, Hu H, Yiu KH, Luo T, Zhou Z, Xu L, Zhang S, Li K, Yu Z.
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120.Jia LQ, Yang GL, Ren L, Chen WN, Feng JY, Cao Y, Zhang L, Li XT, Lei P.
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   Enhanced in vitro antitumor efficacy and strong anti-cell-migration activity of a
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122.Chen Y, Liu J, Yuan B, Cao C, Qin S, Cao X, Bian G, Wang Z, Jiang J.
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126.Deng Z, Yan H, Hu J, Zhang S, Peng P, Liu Q, Guo D.
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127.Zhao WH, Gou BD, Zhang TL, Wang K.
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128.Guo W, Liu W, Hong S, Liu H, Qian C, Shen Y, Wu X, Sun Y, Xu Q.
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129.Li JZ, Yu SY, Wu JH, Shao QR, Dong XM.
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