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Non-radioactive LATS in vitro Kinase Assay
非放射法体外测定LATs激酶活性   

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Abstract

This protocol describes a method to directly measure LATS activity by an in vitro kinase assay using YAP as a substrate.

Keywords: LATS(LATS(大肿瘤抑制因子)), Phosphorylation(磷酸化), in vitro kinase assay(体外激酶活性检测), Hippo pathway(Hippo信号通路), YAP(YAP)

Background

Large tumor suppressor 1/2 (LATS1/2) are protein kinases and core components of the Hippo pathway, which regulates organ size and tissue homeostasis. LATS kinase is activated by phosphorylation on its hydrophobic motif (HM, Thr 1079 for LATS1 and Thr 1041 for LATS2). As a result, Western blotting with phosphoantibody recognizing LATS at HM provides an indirect way to assess LATS kinase activity (Data analysis, Figure 1). In addition, active LATS phosphorylates and inhibits the transcription co-activator Yes-associated protein (YAP) at Ser 127, leading to YAP binding to 14-3-3 and cytoplasmic retention (Zhao et al., 2007). Using YAP as a substrate in LATS in vitro kinase assay provides a method to directly assess LATS kinase activity. Through this assay, we were able to show that serum starvation and sorbitol-induced osmotic activate LATS (Yu et al., 2012; Hong et al., 2017) and further lead to YAP Ser 127 phosphorylation (Data analysis, Figure 2).

Materials and Reagents

  1. Pipette tips
  2. 10 cm plates
  3. 1.5 ml Eppendorf tube
  4. Dialyzer (EMD Millipore, catalog number: 71507-3 )
  5. pGEX-KG-GST-YAP plasmid (Addgene, catalog number: 33052 )
  6. BL21(DE3) competent cells (Agilent Technologies, catalog number: 230134 )
  7. HEK293A cells
  8. LB broth (Fisher Scientific, catalog number: BP1426-2 )
  9. Carbenicillin disodium salt (Sigma-Aldrich, catalog number: 205805-250MG )
  10. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, catalog number: I5502 )
  11. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010049 )
  12. Protease inhibitor cocktail tablet (Roche Diagnostics, catalog number: 11873580001 )
  13. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626-5G )
  14. Dithiothreitol (DTT) (Bio-Rad Laboratories, catalog number: 1610611 )
  15. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  16. Glutathione Sepharose 4B (GE Healthcare, catalog number: 17075601 )
  17. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
  18. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
  19. Phosphatase inhibitor mini tablet (Thermo Fisher Scientific, Thermo ScientificTM , catalog number: 88667 )
  20. Protein A/G magnetic beads (Thermo Fisher Scientific, Thermo ScientificTM , catalog number: 88802 )
  21. Kinase buffer (New England Biolabs, catalog number: B6022S )
  22. Cold ATP (Sigma-Aldrich, catalog number: A2383 )
  23. Sorbitol (Fisher Scientific, catalog number: S459-500 )
  24. Antibodies
    1. Lats1 antibody (Cell Signaling Technology, catalog number: 3477S )
    2. pYAP S127 antibody (Cell Signaling Technology, catalog number: 4911S )
    3. pLATS-HM antibody (Cell Signaling Technology, catalog number: 8654S )
    4. GAPDH antibody (Santa Cruz Biotechnology, catalog number: sc-25778 )
    5. GST antibody (Sigma-Aldrich, catalog number: SAB4200237 )
  25. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3294 )
  26. Tris-base (Fisher Scientific, catalog number: BP152-10 )
  27. L-glutathione reduced (Sigma-Aldrich, catalog number: G4251 )
  28. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  29. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-10 )
  30. Glycerol (Fisher Scientific, catalog number: G33-1 )
  31. Sodium lauryl sulfate (SDS) (Fisher Scientific, catalog number: S529-500 )
  32. Bromophenol blue (Bio-Rad Laboratories, catalog number: 1610404 )
  33. Sodium fluoride (NaF) (Acros Organics, catalog number: 424325000 )
  34. EDTA (Mediatech, catalog number: 46-034-CI )
  35. NP-40 substitute (Sigma-Aldrich, catalog number: 74385 )
  36. Elution buffer (see Recipes)
  37. Dialysis buffer (see Recipes)
  38. 4x SDS sample buffer (see Recipes)
  39. Mild lysis buffer (see Recipes)
  40. TBS buffer (see Recipes)

Equipment

  1. Pipettes
  2. Spectrophotometer (Biochrom, model: Biochrom WPA Biowave II )
  3. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM , model: SorvallTM RC 6 Plus)
  4. Sonic dismembrator (Sonicator) (Fisher Scientific, model: Model 505 )
  5. Bench-top centrifuge (Denville Scientific, model: Denville 300D , catalog number: C0265-24)
  6. Magnetic rack (Thermo Fisher Scientific, catalog number: 12321D )
  7. Shaker (Eppendorf, New BrunswickTM, model: Excella® E24 )
  8. Heat block

Procedure

  1. Purification of GST-YAP protein
    1. Transform pGEX-KG-GST-YAP in BL21(DE3) competent cells by heat shock at 42 °C for 45 sec.
    2. Plate the cells on LB agar plates with 100 μg/ml carbenicillin and incubate overnight at 37 °C.
    3. Pick a single colony and inoculate in 5 ml LB broth with carbenicillin. Incubate overnight at 37 °C.
    4. Pour the 5 ml cultured bacteria in 500 ml LB broth and grow at 37 °C for around 2-3 h until OD600 = 0.6-0.8.
    5. Induce with 0.1-0.4 mM IPTG. Shake overnight at 16 °C.
    6. Spin down the bacteria at 4,000 x g for 15 min at 4 °C.
    7. Lyse the cells in 9 ml of PBS + 1x protease inhibitor + 1 mM PMSF + 1 mM DTT.
    8. Sonicate on ice. Total sonication time 1 min 30 sec, interval 15 sec. Amp = 30%.
    9. Add 1% Triton-X to the sonicated bacterial samples. Shake in 4 °C for 20-30 min.
    10. At the meantime, wash the glutathione Sepharose 4B beads 3 times with 0.1% Triton-X in PBS. 200 μl beads were used for 500 ml of bacteria culture.
    11. Spin down the bacteria at 10,000 x g for 30 min at 4 °C.
    12. Collect supernatant and add the beads. Rotate overnight at 4 °C.
    13. Centrifuge down the beads at 500 x g for 5 min at 4 °C.
    14. Wash with PBS + 0.1% Triton-X 3 times, 5 min each.
    15. Add 500 μl elution buffer and rotate for 1 h at 4 °C.
    16. Spin down the sample at 500 x g for 5 min at 4 °C.
    17. Repeat elution again.
    18. Collect the elution for dialysis against dialysis buffer at 4 °C twice.
    19. Snap freeze the protein and store at -80 °C

  2. LATS in vitro kinase assay
    1. HEK293A cells are cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS).
    2. Cells were seeded in 10 cm plates at a density of 1.5 x 106 per plate and were incubated in 37 °C, 5% CO2 until 80% confluent. A positive control can be cells that are serum starved for 30 min (Figure 2).
    3. Wash the cells with cold PBS.
    4. Remove PBS and scrape down the cells with 1 ml cold Mild lysis buffer (see Recipes) supplemented with 1x protease inhibitor, 1x phosphatase inhibitor, and 1 mM PMSF. Transfer the cells into 1.5 ml Eppendorf tubes.
    5. Incubate the samples on ice for 10 min.
    6. Spin down the lysates at 12,000 x g for 15 min at 4 °C.
    7. Collect the supernatants and incubate with 1 μl LATS1 antibody per sample overnight at 4 °C with constant rotation.
    8. Using a magnetic rack, wash the magnetic beads (10 μl per sample) 3 times with Mild lysis buffer. Resuspend the magnetic beads in 50 μl of Mild lysis buffer and add to the lysate. Rotate for 1 h at 4 °C.
    9. Using a magnetic rack, wash samples 3 times (10 min each) with 1 ml Mild lysis buffer per sample at 4 °C.
    10. Wash samples once with 1 ml 1x TBS (see Recipes) per sample for 10 min at 4 °C.
    11. While washing with TBS, prepare a master mix for the kinase reaction. Each kinase reaction contains 4 μl 10x kinase buffer, 500 μM cold ATP, and 1 μg GST-YAP in 40 μl reaction mix.
    12. Spin down the samples and remove the TBS using a magnetic rack. Add 40 μl of master mix into each reaction.
    13. Shake the reaction mixture at 30 °C for 30 min.
    14. Add 4x SDS sample buffer to stop the reaction, and heat the samples at 100 °C for 5 min.
    15. The samples are then subjected to SDS-PAGE and Western blotting.

Data analysis

Sorbitol or serum starvation-induced LATS activity can be assayed by detecting the phosphorylation of LATS at HM (Figure 1). Direct kinase activity of LATS can be measured by detecting the phosphorylation level of YAP in LATS in vitro kinase assay (Figure 2). Increased YAP phosphorylation represents increased LATS kinase activity.


Figure 1. Serum starvation and sorbitol-induced osmotic stress induce LATS HM phosphorylation. HEK293A cells were treated with 0.2 M sorbitol for 30 or 60 min in the presence or absence of serum. LATS phosphorylation at the hydrophobic motif (HM) was detected with the phospho-specific pLATS antibody. (pLATS-HM Ab 1:1,000 dilution, LATS1 Ab 1:2,000 dilution, GAPDH Ab 1:2,000 dilution in 5% BSA containing TBST).


Figure 2. Kinase activity measured by LATS in vitro kinase assay. HEK293A cells were serum starved or treated with 0.2 M sorbitol for 30 min. LATS1 was immunoprecipitated and an in vitro kinase assay was performed using recombinant GST-YAP as a substrate. Phosphorylation of YAP was determined by immunoblotting with a phospho-YAP (S127) antibody. (pYAP S127 Ab 1:1,000 dilution, GST Ab 1:2,000 dilution, LATS1 Ab 1:2,000 dilution in 5% BSA containing TBST).

Recipes

  1. Elution buffer
    50 mM Tris pH 8.0
    20 mM glutathione
    Note: Prepared fresh.
  2. Dialysis buffer
    20 mM Tris pH 8.0
    2 mM β-mercaptoethanol
    100 mM NaCl
    10% glycerol
    Note: Prepared fresh.
  3. 4x SDS sample buffer
    200 mM Tris pH 6.8
    8% SDS
    0.1% bromophenol blue
    40% glycerol
    20% β-mercaptoethanol
    Note: Stored at room temperature.
  4. Mild lysis buffer
    20 mM Tris pH 7.5
    100 mM NaCl
    50 mM NaF
    2 mM EDTA
    1% NP-40 substitute
    Note: Stored at 4 °C.
  5. TBS buffer
    20 mM Tris-HCl pH 7.5
    170 mM NaCl
    Note: Stored at room temperature.

Acknowledgments

This protocol was adapted from and used in Yu et al. (2012) and Hong et al. (2017). A.W.H. is supported in part by the T32 GM007752 training grant. K.L.G. is supported by grants from National Institute of Health ( CA196878, GM051586)

References

  1. Hong, A. W., Meng, Z., Yuan, H. X., Plouffe, S. W., Moon, S., Kim, W., Jho, E. H. and Guan, K. L. (2017). Osmotic stress-induced phosphorylation by NLK at Ser128 activates YAP. EMBO Rep 18(1): 72-86.
  2. Yu, F. X., Zhao, B., Panupinthu, N., Jewell, J. L., Lian, I., Wang, L. H., Zhao, J., Yuan, H., Tumaneng, K., Li, H., Fu, X. D., Mills, G. B. and Guan, K. L. (2012). Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling. Cell 150(4): 780-791.
  3. Zhao, B., Wei, X., Li, W., Udan, R. S., Yang, Q., Kim, J., Xie, J., Ikenoue, T., Yu, J., Li, L., Zheng, P., Ye, K., Chinnaiyan, A., Halder, G., Lai, Z. C. and Guan, K. L. (2007). Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev 21(21): 2747-2761.

简介

该方案描述了通过使用YAP作为底物的体外激酶测定直接测量LATS活性的方法。
【背景】大肿瘤抑制剂1/2(LATS1 / 2)是河马途径的蛋白激酶和核心成分,其调节器官大小和组织稳态。 LATS激酶通过其疏水基序上的磷酸化被激活(HM,对于LATS1为Thr 1079,对于LATS2为Thr 1041)。 结果,在HM中识别LATS的抗体抗体的Western印迹提供了评估LATS激酶活性的间接方法(Data analysis,图1)。 此外,活性LATS在Ser 127磷酸化并抑制转录共激活物是相关蛋白(YAP),导致YAP结合到14-3-3和细胞质保留(Zhao等人,2007)。 在LATS体外激酶测定中使用YAP作为底物提供了直接评估LATS激酶活性的方法。 通过该测定,我们能够显示血清饥饿和山梨醇诱导的渗透激活LATS(Yu等人,2012; Hong等人,2017)和进一步的 导致YAP Ser 127磷酸化(数据分析,图2)。

关键字:LATS(大肿瘤抑制因子), 磷酸化, 体外激酶活性检测, Hippo信号通路, YAP

材料和试剂

  1. 移液器提示
  2. 10厘米板
  3. 1.5 ml Eppendorf管
  4. 透析器(EMD Millipore,目录号:71507-3)
  5. pGEX-KG-GST-YAP质粒(Addgene,目录号:33052)
  6. BL21(DE3)感受态细胞(Agilent Technologies,目录号:230134)
  7. HEK293A细胞
  8. LB肉汤(Fisher Scientific,目录号:BP1426-2)
  9. 羧苄青霉素二钠盐(Sigma-Aldrich,目录号:205805-250MG)
  10. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Sigma-Aldrich,目录号:I5502)
  11. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010049)
  12. 蛋白酶抑制剂混合片(Roche Diagnostics,目录号:11873580001)
  13. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626-5G)
  14. 二硫苏糖醇(DTT)(Bio-Rad Laboratories,目录号:1610611)
  15. Triton X-100(Sigma-Aldrich,目录号:T9284)
  16. 谷胱甘肽琼脂糖4B(GE Healthcare,目录号:17075601)
  17. Dulbecco改良Eagle's培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:11965092)
  18. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10437028)
  19. 磷酸酶抑制剂迷你片(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88667)
  20. 蛋白A / G磁珠(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88802)
  21. 激酶缓冲液(New England Biolabs,目录号:B6022S)
  22. 冷ATP(Sigma-Aldrich,目录号:A2383)
  23. 山梨醇(Fisher Scientific,目录号:S459-500)
  24. 抗体
    1. Lats1抗体(Cell Signaling Technology,目录号:3477S)
    2. pYAP S127抗体(Cell Signaling Technology,目录号:4911S)
    3. pLATS-HM抗体(Cell Signaling Technology,目录号:8654S)
    4. GAPDH抗体(Santa Cruz Biotechnology,目录号:sc-25778)
    5. GST抗体(Sigma-Aldrich,目录号:SAB4200237)
  25. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3294)
  26. Tris-base(Fisher Scientific,目录号:BP152-10)
  27. L-谷胱甘肽还原(Sigma-Aldrich,目录号:G4251)
  28. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  29. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-10)
  30. 甘油(Fisher Scientific,目录号:G33-1)
  31. 十二烷基硫酸钠(SDS)(Fisher Scientific,目录号:S529-500)
  32. 溴酚蓝(Bio-Rad Laboratories,目录号:1610404)
  33. 氟化钠(NaF)(Acros Organics,目录号:424325000)
  34. EDTA(Mediatech,目录号:46-034-CI)
  35. NP-40替代品(Sigma-Aldrich,目录号:74385)
  36. 洗脱缓冲液(见配方)
  37. 透析缓冲液(见配方)
  38. 4x SDS样品缓冲液(参见食谱)
  39. 轻度溶解缓冲液(参见食谱)
  40. TBS缓冲(见配方)

设备

  1. 移液器
  2. 分光光度计(Biochrom,型号:Biochrom WPA Biowave II)
  3. 离心机(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Sorvall TM RC 6 Plus)
  4. 声波除垢器(Sonicator)(Fisher Scientific,型号:505型)
  5. 台式离心机(Denville Scientific,型号:Denville 300D,目录号:C0265-24)
  6. 磁性架(Thermo Fisher Scientific,目录号:12321D)
  7. Shaker(Eppendorf,New Brunswick TM ,型号:Excella ® E24)
  8. 热块

程序

  1. 纯化GST-YAP蛋白
    1. 通过在42℃热休克45秒,在BL21(DE3)感受态细胞中转化pGEX-KG-GST-YAP。
    2. 用100μg/ ml羧苄青霉素在LB琼脂平板上培养细胞,并在37℃下孵育过夜。
    3. 挑取单个菌落,并接种于含有羧苄青霉素的5ml LB肉汤中。在37°C孵育过夜。
    4. 将5ml培养的细菌倒入500ml LB肉汤中,并在37℃下生长2-3小时,直到OD 600 = 0.6-0.8。
    5. 诱导0.1-0.4 mM IPTG。在16°C过夜振荡。
    6. 在4℃下将细菌以4,000 x g 旋转15分钟。
    7. 将细胞溶解在9ml PBS + 1x蛋白酶抑制剂+ 1mM PMSF + 1mM DTT中
    8. 在冰上超声处理总超声处理时间1分30秒,间隔15秒。 Amp = 30%。
    9. 向超声处理的细菌样品中加入1%Triton-X。在4℃下摇匀20-30分钟
    10. 同时,用0.1%Triton-X在PBS中洗涤谷胱甘肽Sepharose 4B珠珠3次。将200μl珠子用于500ml细菌培养。
    11. 在4℃下以10,000×g / min旋转细菌30分钟。
    12. 收集上清液并加入珠粒。在4℃下旋转过夜。
    13. 在4℃下以500×g离心珠离心5分钟。
    14. 用PBS + 0.1%Triton-X洗3次,每次5分钟。
    15. 加入500μl洗脱缓冲液,4°C旋转1 h。
    16. 在4℃下将样品在500℃下旋转5分钟。
    17. 再次重洗。
    18. 在4℃下收集透析缓冲液的洗脱液两次。
    19. 快速冻结蛋白质并储存于-80°C
  2. LATS体外激酶测定
    1. 将HEK293A细胞在含有10%胎牛血清(FBS)的Dulbecco's改良的Eagle培养基(DMEM)中培养。
    2. 将细胞以每板1.5×10 6的密度接种在10cm平板中,并在37℃,5%CO 2/2下孵育至80%汇合。阳性对照可以是血清饥饿30分钟的细胞(图2)
    3. 用冷PBS洗涤细胞。
    4. 除去PBS,并用1ml冷的轻度裂解缓冲液(参见Recipes),补充有1x蛋白酶抑制剂,1×磷酸酶抑制剂和1mM PMSF来清洗细胞。将细胞转移到1.5ml Eppendorf管中。
    5. 将样品在冰上孵育10分钟。
    6. 在4℃下将裂解物以12,000xg旋转15分钟。
    7. 收集上清液,每个样品1μlLATS1抗体在4°C恒定旋转过夜孵育过夜。
    8. 使用磁性架,用轻度裂解缓冲液洗涤磁珠(每个样品10μl)3次。将磁珠重悬于50μl轻度裂解缓冲液中,并加入裂解液中。在4°C旋转1小时。
    9. 使用磁性架,在4℃下,每个样品洗1次(每次10分钟),每次1ml温和裂解缓冲液。
    10. 在4℃下,每个样品用1ml 1x TBS(参见食谱)洗涤样品一次10分钟。
    11. 在用TBS洗涤的同时,制备激酶反应的主混合物。每个激酶反应在40μl反应混合物中含有4μl10x激酶缓冲液,500μM冷ATP和1μgGST-YAP。
    12. 旋转样品并使用磁性架拆除TBS。在每个反应中加入40μl的主混合物。
    13. 将反应混合物在30℃下摇动30分钟。
    14. 加入4x SDS样品缓冲液以停止反应,并将样品在100℃下加热5分钟。
    15. 然后将样品进行SDS-PAGE和Western印迹。

数据分析

山梨醇或血清饥饿诱导的LATS活性可以通过检测HM的LATS的磷酸化来测定(图1)。 LATS的直接激酶活性可以通过检测LATS体外激酶测定中YAP的磷酸化水平来测量(图2)。增加的YAP磷酸化代表增加的LATS激酶活性

图1.血清饥饿和山梨醇诱导的渗透胁迫诱导LATS HM磷酸化。 HEK293A细胞在存在或不存在血清的情况下用0.2M山梨糖醇处理30或60分钟。用磷酸特异性pLATS抗体检测疏水基序(HM)处的LATS磷酸化。 (pLATS-HM Ab 1:1,000稀释,LATS1 Ab 1:2,000稀释,含有TBST的5%BSA中的GAPDH Ab 1:2,000稀释度)。


图2.通过LATS体外激酶测定测定的激酶活性。 HEK293A细胞血清饥饿或用0.2M山梨糖醇处理30分钟。 LATS1被免疫沉淀,并使用重组GST-YAP作为底物进行体外激酶测定。通过用磷酸-YAP(S127)抗体的免疫印迹测定YAP的磷酸化。 (pYAP S127 Ab 1:1,000稀释,GST Ab 1:2,000稀释,LATS1 Ab 1:2,000稀释于含有TBST的5%BSA中)。

食谱

  1. 洗脱缓冲液
    50mM Tris pH 8.0
    20 mM谷胱甘肽
    注意:准备新鲜。
  2. 透析缓冲液
    20 mM Tris pH 8.0
    2mMβ-巯基乙醇
    100 mM NaCl
    10%甘油
    注意:准备新鲜。
  3. 4x SDS样品缓冲液
    200 mM Tris pH 6.8
    8%SDS
    0.1%溴酚蓝
    40%甘油
    20%β-巯基乙醇
    注意:在室温下保存。
  4. 轻度溶解缓冲液
    20mM Tris pH7.5
    100 mM NaCl
    50 mM NaF
    2 mM EDTA
    1%NP-40替代品
    注意:存储在4°C。
  5. TBS缓冲区
    20mM Tris-HCl pH 7.5
    170 mM NaCl
    注意:在室温下保存。

致谢

这个协议是从Yu等人改编而来的。 (2012)和Hong等人(2017)。 A.W.H.部分由T32 GM007752培训补助金支持。 K.L.G.得到了国家卫生研究院的资助(CA196878,GM051586)

参考

  1. Hong,AW,Meng,Z.,Yuan,HX,Plouffe,SW,Moon,S.,Kim,W.,Jho,EH and Guan,KL(2017)。  通过NLK在Ser128的渗透应激诱导的磷酸化激活YAP。 EMBO Rep 18(1):72-86。
  2. Yu,FX,Zhao,B.,Panupinthu,N.,Jewell,JL,Lian,I.,Wang,LH,Zhao,J.,Yuan,H.,Tumaneng,K.,Li,H.,Fu,XD ,Mills,GB and Guan,KL(2012)。  通过G蛋白偶联受体信号调节Hippo-YAP途径。 150(4):780-791。
  3. Zhao,B.,Wei,X.,Li,W.,Udan,RS,Yang,Q.,Kim,J.,Xie,J.,Ikenoue,T.,Yu,J.,Li,L.,Zheng ,P.,Ye,K.,Chinnaiyan,A.,Halder,G.,Lai,ZC and Guan,KL(2007)。< a class =“ke-insertfile”href =“http: ncbi.nlm.nih.gov/pubmed/17974916“target =”_ blank“>通过Hippo途径失活YAP癌蛋白参与细胞接触抑制和组织生长控制。 Genes Dev 21(21):2747-2761。
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引用:Hong, A. W. and Guan, K. (2017). Non-radioactive LATS in vitro Kinase Assay. Bio-protocol 7(14): e2391. DOI: 10.21769/BioProtoc.2391.
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