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Phosphatase Protection Assay: 14-3-3 Binding Protects the Phosphate group of RSG from λ Protein Phosphatase
磷酸酶保护试验:14-3-3结合保护λ蛋白磷酸酶中的RSG磷酸根   

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Abstract

14-3-3 proteins regulate diverse cellular processes in eukaryotes by binding to phospho-serine or threonine of target proteins. One of the physiological functions of 14-3-3 is to bind and protect phosphate groups of the target proteins against phosphatases. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in the feedback regulation of biosynthetic genes of plant hormone gibberellin. 14-3-3 binds to phospho-Ser-114 in RSG. Ca2+-dependent protein kinase NtCDPK1 was identified as a kinase that phosphorylates Ser-114 of RSG. Our recent study revealed that NtCDPK1 forms a heterotrimer with RSG and 14-3-3 and that 14-3-3 was transferred from NtCDPK1 to phosphorylated RSG (Ito et al., 2014). In the course of the study, we found that 14-3-3 protects the phosphate group of RSG from λ protein phosphatase in vitro. Here, we describe a protocol for in vitro phosphatase protection assay. To detect the phosphorylation state of proteins, we used Phos-tag SDS-PAGE and autoradiography. This protocol can be adapted for the examinations whether the phosphoprotein-binding proteins protect the phosphate group of target proteins from phosphatases although protein kinases may be required for the phosphorylation of target proteins.

Keywords: Phosphatase(磷酸酶), 14-3-3(14-3-3), Phosphorylation(磷酸化), Protection(保护), Phosphate(磷酸盐)

Materials and Reagents

  1. 20 mM ATP
  2. [γ-32P] ATP (5,000 Ci/mmol, 10 mCi/ml) (Institute of Isotopes, catalog number: SBP-401
  3. λ protein phosphatase (New England Biolabs, catalog number: P0753S )
  4. Amylose resin (New England Biolabs, catalog number: E8021 )
  5. COSMOGEL His-Accept (Nacalai Tesque, catalog number: 08368-12 )
  6. Glutathione Sepharose 4B (GE Healthcare, catalog number: 17-0756-01 )
  7. Phos-tag acrylamide (Wako Pure Chemical Industries, catalog number: 304-93521 )
  8. Wide-view prestained protein size marker III (Wako Pure Chemical Industries, catalog number: 230-02461 )
  9. His.tag monoclonal antibody (Merck Millipore, catalog number: 70796-4CN )
  10. Phenylmethanesulfonyl fluoride (PMSF)
  11. Dialysis buffer (see Recipes)
  12. 10x phosphorylation buffer (see Recipes)
  13. Binding buffer (see Recipes)
  14. Dephosphorylation buffer (see Recipes)
  15. 2x SDS protein sample buffer (see Recipes)

Equipment

  1. 1.5 ml microcentrifuge tubes
  2. Refrigerated microcentrifuge
  3. Microtube rotator
  4. GELoader tips 0.5-20 µl (Eppendorf, catalog number: 0030 001.222 )
  5. Heat block
  6. SDS-PAGE apparatus
  7. Gel transfer apparatus
  8. Power supply
  9. Shaker
  10. Phosphorimager
  11. Chemiluminescence imaging system

Procedure

  1. Preparation of recombinant proteins
    1. MBP-RSG, His-14-3-3 and GST-NtCDPK1 were expressed in Escherichia coli containing pMALc2-RSG, pET16b-14-3-3 or pGEX 4T-1-NtCDPK1, respectively, as described in the references (Ishida et al., 2008; Ito et al., 2010; Ito et al., 2014).
    2. MBP-RSG, His-14-3-3 and GST-NtCDPK1 were purified by using Amylose resin, COSMOGEL His-Accept or Glutathione Sepharose 4B according to the manual (pMALTM Protein Fusion & Purification System, Ni-NTA His Bind Resins, or GST gene fusion system handbook, respectively).
    3. The purified proteins were dialyzed separately overnight against a 500-1,000-fold volume of ice-cold dialysis buffer.
    4. Glycerol, corresponding to 40% by volume of the dialyzed protein solutions, was added to the dialyzed protein solutions to make approximately 50% (v/v) glycerol (final concentration), and the protein solutions were stored at -20 °C.
      Note: Dialyzed protein solution contains approximately 30% (v/v) glycerol.

  2. Phosphorylation of RSG by NtCDPK1
    1. Prepare reaction mixtures in each of 1.5 ml microcentrifuge tube using the following:
      5 µl 10x phosphorylation buffer
      5 µl 1% (v/v) β-mercaptoethanol (dilute immediately before use)
      2.5 µl 20 mM ATP with or without 1 µl [γ-32P] ATP (5,000 µCi/mmol, 10 mCi/ml)
      2 µg MBP-RSG
      100 ng GST-NtCDPK1
      H2O up to 50 µl
    2. Mix and incubate the reaction mixture for 1 h at 30 °C.

  3. 14-3-3 binding to phosphorylated RSG
    1. Gently invert the container of amylose resin to resuspend the slurry.
    2. Transfer 30 µl of resuspended slurry to each of 1.5 ml microcentrifuge tube.
    3. Add 1 ml of ice-cold binding buffer to each microcentrifuge tube to equilibrate the resin for use.
    4. Spin down the resin at 1,000 x g for a few seconds at 4 °C.
    5. Carefully remove and discard the supernatant.
    6. Repeat steps C3-5 two more times for a total of three washes.
    7. Add 250 µl of ice-cold binding buffer, all (50 µl) of the reaction mixture phosphorylated RSG and 3 µg His-14-3-3 to the resin.
    8. Gently mix the mixture for 30 min at 4 °C on a microtube rotator to bind MBP-RSG to the amylose resin and His-14-3-3.
    9. Spin down the resin at 1,000 x g for a few seconds at 4 °C.
    10. Carefully remove and discard the supernatant.
    11. Wash the resin with 1 ml of ice-cold binding buffer to remove ATP and unbound proteins.
    12. Gently mix by inverting the tube several times.
    13. Spin down the resin at 1,000 x g for a few seconds at 4 °C.
    14. Carefully remove and discard the supernatant.
    15. Repeat steps C11-14 three more times for a total of four washes.
    16. Completely remove the supernatant by using a micropipettor equipped with GELoader Tips.

  4. Dephosphorylation of RSG by λ protein phosphatase
    1. Add 200 µl of ice-cold dephosphorylation buffer and 1 µl (400 U) of λ protein phosphatase to the resin.
    2. Gently mix the mixture for 30 min at 4 °C on a microtube rotator.
    3. Wash unbound proteins from the resin with 1 ml of ice-cold binding buffer.
    4. Gently mix by inverting the tube several times.
    5. Spin down the resin at 1,000 x g for a few seconds at 4 °C.
    6. Carefully remove and discard the supernatant.
    7. Repeat steps D3-6 three more times for a total of four washes.
    8. Completely remove the supernatant by using a micropipettor equipped with GELoader tips.

  5. Detection of phosphorylation state of RSG
    1. Add 60 µl of 2x SDS protein sample buffer to the resin.
    2. Heat the tubes for 5 min in a 95 °C heat block to denature the proteins.
    3. Load 25 µl of the supernatant and 3 µl of protein size marker and perform Phos-tag SDS-PAGE (Kinoshita and Kinoshita-Kikuta, 2011) for the detection of MBP-RSG phosphorylated with cold ATP or normal SDS-PAGE for the detection of MBP-RSG phosphorylated with [γ-32P] ATP.
    4. Perform coomassie brilliant blue (CBB) staining of Phos-tag SDS-PAGE gel to examine the phosphorylation state of MBP-RSG phosphorylated with cold ATP.
    5. To detect the phosphorylation state of MBP-RSG phosphorylated with [γ-32P] ATP, dry the SDS-PAGE gel and place an imaging plate over the dried gel. Expose overnight to visualize the radioactive signals. Scan the imaging plate by using an imaging system, Typhoon FLA 9500.
    6. Perform immunoblot analysis with anti-14-3-3 (Igarashi et al., 2001) antibody or anti-His-tag monoclonal antibody for the detection of His-14-3-3 bound to MBP-RSG by using an imaging system, ImageQuant LAS 4000.

Representative data



Figure 1. The phosphorylation state of Ser-114 in RSG is protected from λ protein phosphatase by 14-3-3. MBP-RSG proteins phosphorylated with cold ATP were resolved on Phos-tag SDS-PAGE and visualized by coomassie brilliant blue (CBB) staining. MBP-RSG proteins phosphorylated with [γ-32P] ATP were resolved on SDS-PAGE and detected by using imaging system. Several amino acids of RSG are phosphorylated by NtCDPK1. When His-14-3-3 does not bind to MBP-RSG, phosphate groups of RSG are completely removed by λ protein phosphatase. On the other hand, when MBP-RSG that bound to His-14-3-3 is treated with λ protein phosphatase, the phosphorylation state of Ser-114 in RSG is protected from λ protein phosphatase. Top, middle and bottom arrowheads represent multiply phosphorylated, Ser-114 phosphorylated and dephosphorylated MBP-RSG, respectively. For more information, please see Ito et al. (2014).

Notes

  1. We used λ protein phosphatase but not bacterial alkaline phosphatase for this assay because 14-3-3 tends to dissociate from RSG under alkaline conditions. If alkaline conditions do not lead to the dissociation of target protein complexes, bacterial alkaline phosphatase can be used for this assay.
  2. The condition for stable protein-protein interactions vary among different proteins. The optimum buffer condition for target protein-protein interactions could be found by changing pH level, NaCl (KCl) concentration and detergent type and concentration.
  3. In case that phosphorylation or dephosphorylation is excessive or insufficient, changing the reaction times or the amounts of kinase and phosphatase could gain satisfactory results.

Recipes

  1. Dialysis buffer
    25 mM Tris-HCl (pH 7.5 at 4 °C)
    150 mM NaCl
    0.1% (v/v) Triton X-100
    30% (v/v) glycerol
    0.05% (v/v) β-mercaptoethanol
    0.1 mM PMSF
    Note: β-mercaptoethanol and PMSF are added immediately before use.
  2. 10x phosphorylation buffer
    200 mM Tris-HCl (pH 7.5 at room temperature)
    100 mM NaCl
    1% (v/v) Triton X-100
    5 mM CaCl2
  3. Binding buffer
    25 mM MOPS-NaOH (pH 7.0 at 4 °C)
    25 mM NaCl
    0.1% (v/v) Triton X-100
    0.05% (v/v) β-mercaptoethanol
    0.1 mM PMSF
    Note: β-mercaptoethanol and PMSF are added immediately before use.
  4. Dephosphorylation buffer
    25 mM MOPS-NaOH (pH 7.0 at 4 °C)
    25 mM NaCl
    0.1% (v/v) Triton X-100
    1 mM MnCl2
    0.05% (v/v) β-mercaptoethanol
    0.1 mM PMSF
    Note: β-mercaptoethanol and PMSF are added immediately before use.
  5. 2x SDS protein sample buffer
    250 mM Tris-HCl (pH 6.8 at room temperature)
    4.0% (w/v) SDS
    20% (v/v) glycerol
    0.02% (w/v) Bromophenol blue
    10% (v/v) β-mercaptoethanol
    Note: β-mercaptoethanol is added immediately before use.

Acknowledgments

This work was supported by the Japan Society of the Promotion of Science (grant no. 23657038 to Y. T.) and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (grant no. 24118004 to Y. T.).

References

  1. Igarashi, D., Ishida, S., Fukazawa, J. and Takahashi, Y. (2001). 14-3-3 proteins regulate intracellular localization of the bZIP transcriptional activator RSG. Plant Cell 13(11): 2483-2497.
  2. Ishida, S., Yuasa, T., Nakata, M. and Takahashi, Y. (2008). A tobacco calcium-dependent protein kinase, CDPK1, regulates the transcription factor REPRESSION OF SHOOT GROWTH in response to gibberellins. Plant Cell 20(12): 3273-3288.
  3. Ito, T., Nakata, M., Fukazawa, J., Ishida, S. and Takahashi, Y. (2010). Alteration of substrate specificity: the variable N-terminal domain of tobacco Ca(2+)-dependent protein kinase is important for substrate recognition. Plant Cell 22(5): 1592-1604.
  4. Ito, T., Nakata, M., Fukazawa, J., Ishida, S. and Takahashi, Y. (2014). Scaffold function of Ca2+-dependent protein kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 transfers 14-3-3 to the substrate REPRESSION OF SHOOT GROWTH after phosphorylation. Plant Physiol 165(4): 1737-1750.
  5. Kinoshita, E. and Kinoshita-Kikuta, E. (2011). Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling. Proteomics 11(2): 319-323.

简介

14-3-3蛋白通过结合靶蛋白的磷酸 - 丝氨酸或苏氨酸来调节真核生物中的多种细胞过程。 14-3-3的生理功能之一是结合和保护靶蛋白的磷酸基团免受磷酸酶。生长生长的表达(RSG)是涉及植物激素赤霉素的生物合成基因的反馈调节的烟草(烟草属)转录因子。 14-3-3结合RSG中的磷酸-Ser-114。 Ca 2+ - 依赖性蛋白激酶NtCDPK1被鉴定为磷酸化RSG的Ser-114的激酶。我们最近的研究揭示NtCDPK1与RSG和14-3-3形成异源三聚体,并且14-3-3从NtCDPK1转移到磷酸化RSG(Ito等人,2014)。在研究过程中,我们发现14-3-3在体外保护RSG的磷酸基团免受λ蛋白磷酸酶的影响。在这里,我们描述了体外磷酸酶保护测定的协议。为了检测蛋白质的磷酸化状态,我们使用Phos-tag SDS-PAGE和放射自显影。该方案可以适用于磷酸蛋白结合蛋白是否保护靶蛋白的磷酸基团免于磷酸酶的检查,尽管蛋白激酶可能是靶蛋白磷酸化所需的。

关键字:磷酸酶, 14-3-3, 磷酸化, 保护, 磷酸盐

材料和试剂

  1. 20 mM ATP
  2. [γ-32 P] ATP(5,000Ci/mmol,10mCi/ml)(同位素研究所,目录号:SBP-401)
  3. λ蛋白磷酸酶(New England Biolabs,目录号:P0753S)
  4. 直链淀粉树脂(New England Biolabs,目录号:E8021)
  5. COSMOGEL His-Accept(Nacalai Tesque,目录号:08368-12)
  6. 谷胱甘肽琼脂糖4B(GE Healthcare,目录号:17-0756-01)
  7. Phos标记丙烯酰胺(Wako Pure Chemical Industries,目录号:304-93521)
  8. 宽视觉预染蛋白质大小标记物III(Wako Pure Chemical Industries,目录号:230-02461)
  9. His 标签单克隆抗体(Merck Millipore,目录号:70796-4CN)
  10. 苯基甲磺酰氟(PMSF)
  11. 透析缓冲液(参见配方)
  12. 10x磷酸化缓冲液(见配方)
  13. 绑定缓冲区(参见配方)
  14. 脱磷酸缓冲液(参见配方)
  15. 2x SDS蛋白样品缓冲液(见配方)

设备

  1. 1.5 ml微量离心管
  2. 冷藏微量离心机
  3. 微量旋转器
  4. GELoader尖端0.5-20μl(Eppendorf,目录号:0030001.222)
  5. 热块
  6. SDS-PAGE装置
  7. 凝胶转移装置
  8. 电源
  9. 振动器
  10. 磷光仪
  11. 化学发光成像系统

程序

  1. 重组蛋白的制备
    1. 在分别含有pMALc2-RSG,pET16b-14-3-3或pGEX 4T-1-NtCDPK1的大肠杆菌中表达MBP-RSG,His-14-3-3和GST-NtCDPK1,   如参考文献中所述(Ishida等人,2008; Ito等人,2010; Ito 等人,2014)。
    2. MBP-RSG,His-14-3-3和GST-NtCDPK1 通过使用直链淀粉树脂,COSMOGEL His-Accept或谷胱甘肽纯化 根据手册的Sepharose 4B(pMAL TM supra蛋白融合& 纯化系统,Ni-NTA His Bind树脂或GST基因融合系统 手册)。
    3. 将纯化的蛋白质分别用500-1,000倍体积的冰冷的透析缓冲液透析过夜
    4. 甘油,相当于40%体积的透析蛋白 溶液,加入到透析的蛋白质溶液中 大约50%(v/v)甘油(最终浓度)和蛋白质 溶液储存在-20℃。
      注意:透析过的蛋白质溶液含有约30%(v/v)甘油。

  2. NtCDPK1对RSG的磷酸化
    1. 使用以下步骤在每个1.5ml微量离心管中制备反应混合物:
      5μl10x磷酸化缓冲液
      5μl1%(v/v)β-巯基乙醇(即将使用前稀释)
      含有或不含有1μl[γ-32 P] ATP(5,000μCi/mmol,10mCi/ml)的2.5μl20mM ATP,
      2μgMBP-RSG
      100 ng GST-NtCDPK1
      H sub 2 O最多50μl
    2. 混合并将反应混合物在30℃下孵育1小时。

  3. 14-3-3与磷酸化RSG结合
    1. 轻轻倒转直链淀粉树脂的容器以重悬浆料
    2. 转移30微升重悬浮浆液到每个1.5毫升微量离心管。
    3. 向每个微量离心管中加入1ml冰冷的结合缓冲液以平衡树脂使用
    4. 在4℃下,将树脂以1,000×g 旋转几秒钟
    5. 小心取出并弃去上清液。
    6. 重复步骤C3-5两次,共三次洗涤。
    7. 加入250μl冰冷的结合缓冲液,所有(50μl)的反应 混合物将RSG和3μgHis-14-3-3磷酸化到树脂上
    8. 在微量管旋转器上在4℃下轻轻混合混合物30分钟,以将MBP-RSG结合到直链淀粉树脂和His-14-3-3上。
    9. 在4℃下将树脂以1,000×g离心几秒钟。
    10. 小心取出并弃去上清液。
    11. 用1ml冰冷的结合缓冲液洗涤树脂以除去ATP和未结合的蛋白质
    12. 通过倒置管子几次轻轻混合。
    13. 在4℃下将树脂以1,000×g离心几秒钟。
    14. 小心取出并弃去上清液。
    15. 重复步骤C11-14三次,共四次洗涤。
    16. 使用配有GELoader Tips的微量移液器完全除去上清液。

  4. RSG由λ蛋白磷酸酶脱磷酸化
    1. 向树脂中加入200μl冰冷的去磷酸化缓冲液和1μl(400U)λ蛋白磷酸酶。
    2. 在微量管旋转器上在4℃下轻轻混合混合物30分钟。
    3. 用1ml冰冷的结合缓冲液从树脂洗涤未结合的蛋白质
    4. 通过倒置管子几次轻轻混合。
    5. 在4℃下,将树脂以1,000×g 旋转几秒钟
    6. 小心取出并弃去上清液。
    7. 重复步骤D3-6三次,共四次洗涤。
    8. 通过使用配有GELoader尖端的微量移液器完全去除上清液。

  5. 检测RSG的磷酸化状态
    1. 向树脂中加入60μl2×SDS蛋白质样品缓冲液
    2. 在95℃加热块中加热管5分钟以使蛋白质变性
    3. 加载25微升的上清液和3微升的蛋白质大小标记和 进行Phos-tag SDS-PAGE(Kinoshita和Kinoshita-Kikuta,2011)   检测用冷的ATP磷酸化的MBP-RSG或正常的SDS-PAGE 用于检测用[γ-32 P] ATP磷酸化的MBP-RSG
    4. 对Phos-tag SDS-PAGE凝胶进行考马斯亮蓝(CBB)染色   以检查用磷酸化的MBP-RSG的磷酸化状态 冷ATP。
    5. 检测MBP-RSG的磷酸化状态 用[γ-32 P] ATP磷酸化,干燥SDS-PAGE凝胶, 成像板在干凝胶上。 暴露一夜,以可视化 放射性信号。 通过使用成像系统扫描成像板, Typhoon FLA 9500.
    6. 用抗14-3-3进行免疫印迹分析 (Igarashi等人,2001)抗体或抗His标签单克隆抗体   通过使用成像检测His-14-3-3结合到MBP-RSG 系统,ImageQuant LAS 4000。

代表数据



图1.RSG中Ser-114的磷酸化状态受λ蛋白磷酸酶14-3-3保护。用冷ATP磷酸化的MBP-RSG蛋白在Phos-tag SDS-PAGE上解析, 通过考马斯观察 亮蓝(CBB)染色。用[γ-32 P] ATP磷酸化的MBP-RSG蛋白在SDS-PAGE上分辨并使用成像系统检测。 RSG的几个氨基酸被NtCDPK1磷酸化。当His-14-3-3不结合MBP-RSG时,RSG的磷酸基团被λ蛋白磷酸酶完全去除。另一方面,当用λ蛋白磷酸酶处理结合His-14-3-3的MBP-RSG时,RSG中的Ser-114的磷酸化状态受到保护而不受λ蛋白磷酸酶的影响。顶部,中间和底部的箭头分别表示多磷酸化,Ser-114磷酸化和去磷酸化的MBP-RSG。有关详情,请参阅Ito 等(2014)。

笔记

  1. 我们使用λ蛋白磷酸酶,但不是细菌碱性磷酸酶用于该测定,因为14-3-3倾向于在碱性条件下从RSG解离。如果碱性条件不导致靶蛋白复合物的解离,细菌碱性磷酸酶可用于该测定
  2. 不同蛋白质之间稳定的蛋白质 - 蛋白质相互作用的条件不同。通过改变pH水平,NaCl(KCl)浓度和洗涤剂类型和浓度可以发现目标蛋白质 - 蛋白质相互作用的最佳缓冲条件。
  3. 在磷酸化或脱磷酸化过量或不足的情况下,改变反应时间或激酶和磷酸酶的量可获得满意的结果。

食谱

  1. 透析缓冲液
    25mM Tris-HCl(pH7.5,4℃) 150mM NaCl 0.1%(v/v)Triton X-100 30%(v/v)甘油 0.05%(v/v)β-巯基乙醇 0.1mM PMSF
    注意:在使用前立即加入β-巯基乙醇和PMSF。
  2. 10x磷酸化缓冲液
    200mM Tris-HCl(室温下pH7.5)
    100 mM NaCl
    1%(v/v)Triton X-100 5mM CaCl 2
  3. 绑定缓冲区
    25mM MOPS-NaOH(pH7.0,4℃) 25mM NaCl 0.1%(v/v)Triton X-100 0.05%(v/v)β-巯基乙醇 0.1mM PMSF
    注意:在使用前立即加入β-巯基乙醇和PMSF。
  4. 脱磷酸化缓冲液
    25mM MOPS-NaOH(pH7.0,4℃) 25mM NaCl 0.1%(v/v)Triton X-100 1mM MnCl 2
    0.05%(v/v)β-巯基乙醇 0.1mM PMSF
    注意:在使用前立即加入β-巯基乙醇和PMSF。
  5. 2x SDS蛋白样品缓冲液
    250mM Tris-HCl(室温下pH6.8)
    4.0%(w/v)SDS
    20%(v/v)甘油 0.02%(w/v)溴酚蓝
    10%(v/v)β-巯基乙醇 注意:在使用前立即加入β-巯基乙醇。

致谢

这项工作得到日本科学促进会(授予日本科学技术协会23657038号)和日本教育,文化,体育,科学和技术部(授予Y。的授权号24118004)的支持。

参考文献

  1. Igarashi,D.,Ishida,S.,Fukazawa,J.and Takahashi,Y。(2001)。 14-3-3蛋白调节bZIP转录激活剂RSG的细胞内定位。 em> Plant Cell 13(11):2483-2497。
  2. Ishida,S.,Yuasa,T.,Nakata,M.and Takahashi,Y。(2008)。 烟草钙依赖性蛋白激酶CDPK1调节转录因子REPRESSION OF SHOOT GROWTH以响应gibberellins。 Plant Cell 20(12):3273-3288。
  3. Ito,T.,Nakata,M.,Fukazawa,J.,Ishida,S.and Takahashi,Y。(2010)。 底物特异性的改变:烟草Ca(2+)依赖性蛋白的可变N末端结构域激酶对底物识别是重要的。植物细胞22(5):1592-1604。
  4. Ito,T.,Nakata,M.,Fukazawa,J.,Ishida,S.and Takahashi,Y。(2014)。 Ca 2 + 依赖性蛋白激酶的支架功能:烟草 Ca 2 + -DEPENDENT PROTEIN KINASE1将14-3-3转移到底物磷酸化后的生长生长代谢植物生理165(4):1737- 1750。
  5. Kinoshita,E。和Kinoshita-Kikuta,E。(2011)。 在中性pH条件下改进的Phos-tag SDS-PAGE,用于高级蛋白磷酸化分析。 Proteomics 11(2):319-323。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ito, T. and Takahashi, Y. (2015). Phosphatase Protection Assay: 14-3-3 Binding Protects the Phosphate group of RSG from λ Protein Phosphatase. Bio-protocol 5(3): e1395. DOI: 10.21769/BioProtoc.1395.
  2. Ito, T., Nakata, M., Fukazawa, J., Ishida, S. and Takahashi, Y. (2014). Scaffold function of Ca2+-dependent protein kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 transfers 14-3-3 to the substrate REPRESSION OF SHOOT GROWTH after phosphorylation. Plant Physiol 165(4): 1737-1750.
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