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p65 Chromatin Immunoprecipitation Protocol
p65染色质免疫共沉淀法

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Abstract

Chromatin Immunoprecipitation (ChIP) is an important procedure that allows you to verify if a certain protein is physically located at a regulatory region. This information, taken together with other procedures such as luciferase assays and EMSAs, will give definitive proof that the query protein is involved in the transcription of a protein. This procedure for p65 ChIP can be adapted to investigate other proteins; just a change of the antibody will suffice.
The transcription factor known as NF-κB is a homo- or hetero-dimer consisting of members of the Rel/NFKB family. The most abundant NF-κB complexes are made of two different proteins, p65 (Rel-A) and p50 (NFKB1). The NF-κB complex is initially inhibited by IκB by direct binding, thus trapping NF-κB in the cytoplasm. After a stimulatory signal, IκB kinase (IKK) phosphorylates IκB, allowing IκB to undergo proteasome-mediated degradation. The degradation of IκB and phosphorylation of p65 by multiple kinases activates NF-κB, allowing it to transport to the nucleus and cause the transcriptional activation of many of its target genes containing κB sites (consensus sequence: gggRNNYYcc, R = purine Y = pyrimidine), such as PUMA, IL-6, and TNF.

Keywords: P65(P65), NFkB(NFκ), ChIP(炸薯条), Promoter(发起人)

Materials and Reagents

  1. HCT116 cell line
  2. Trypsin (0.05%) (Life Technologies, catalog number: 25300-054 )
  3. DMSO
  4. PBS
  5. Formaldehyde (J.T. Baker, catalog number: 2106-01 )
  6. 1 M Glycine
  7. Liquid nitrogen or a dry ice/100% ethanol
  8. NP-40
  9. Protease inhibitor cocktail tablet (F. Hoffmann-La Roche, catalog number: 04-693159-001 )
  10. p65 antibody (Santa Cruz, catalog number: sc-109 )
  11. Chromatin Immunoprecipitation Assay Kit (EMD Millipore, catalog number: 17-295) online at: http://www.millipore.com/coa.nsf/a73664f9f981af8c852569b9005b4eee/20c0dc520d2b30f0852573be007ffdae/$FILE/17-295-DAM1411265.pdf
  12. Protein A/G PLUS-agarose (has been preblocked with BSA) (Santa Cruz, catalog number: sc-2003 )
  13. Phenol
  14. Chloroform
  15. Glycogen
  16. 70% ethanol
  17. SDS
  18. Triton X-100
  19. EDTA
  20. Tris-HCl
  21. NaCl
  22. NaHCO3
  23. EBC buffer (see Recipes)
  24. ChIP dilution buffer (see Recipes)
  25. 10x protease inhibitor solution (see Recipes)
  26. Low salt immune complex wash buffer (see Recipes)
  27. High salt immune complex wash buffer (see Recipes)
  28. LiCl immune complex wash buffer (see Recipes)
  29. TE buffer (see Recipes)
  30. Elution buffer (see Recipes)

Equipment

  1. Sonicator (Branson Digital Sonifier 450) (Branson Ultrasonics Corp)
  2. Centrifuge
  3. 15 ml conical tubes
  4. T75 flasks
  5. Cell scrapers

Procedure

Outline

  1. Three days before starting this procedure, usually on a Friday, split your cells 1:4 so that they will be confluent on Monday.
  2. Day 1 (Monday)-split your cells.
  3. Day 2 (Tuesday)-treat your cells with whatever compound you have found that causes transcriptional activation of your gene of interest. Fix cells when you believe p65 will be at the regulatory region of your gene of interest. Count cells and aliquot 4 million cells per 15 ml conical tube.
  4. Day 3 (Wednesday)-Sonicate samples, pre-clear samples, and IP with p65 antibody overnight.
  5. Day 4 (Thursday)-Wash samples, elute, and reverse crosslink.
  6. Day 5 (Friday)-Do phenol/chloroform extraction and ethanol precipitation. Proceed to do PCR of your region of interest if you have time that day.

Note: First determine the sonication conditions for your particular cell type to yield DNA fragments between 300-1,000 bp. This is usually around 12% output, 5 cycles of 10 sec each with a 30 sec cooling period in between sonications. A 1.2% agarose Ethidium bromide gel of the DNA will show a brighter smear in this region, as shown in the second lane of Figure 1.



Figure 1. Determining sonication conditions for HCT116 for ChIP. The lane denoted with a star shows the proper sonication conditions.

Day 1

Split your cells. Cells should be in log phase of growth. Usually I use a T75 flask to grow the cells. The cells should be about 60-70% confluent for the next day. If you split a confluent T75 flask of your cells with 3 ml trypsin + 9 ml media, you will need 4 ml cells (~5 x 106 cells) for each T75 (you can do 3 total, so untreated, treated, and a counting dish per T75 flask). These conditions are for HCT116, so adjust accordingly for your cell line.

Day 2

  1. Treat your cells the next day with DMSO or PBS for control (depending on your compound’s solubility) and the compound of interest. To detect p65 on the PUMA promoter after sorafenib treatment, I did an 8 h time point. This may change depending on the transcription rate of your protein. I would use the time that you first started seeing your protein expression increase.
  2. While you wait for your time point, count the cells in your extra flask. This will tell you how many cells/ml you have in your other flasks. If you can’t do this due to limited sample amounts, after the time point for your compound, and before you fix the cells, count them.
  3. Add formaldehyde to a final concentration of 1% in your flask. Let sit at room temperature (RT) with gentle rocking for 15 min.
  4. Add 1 M glycine to a final concentration of 125 mM to stop the reaction. Mix gently.
  5. Using a cell scraper, scrape off the cells and spin down cells at 400 x g for 5 min at 4 °C. Aspirate supernatant.
  6. Resuspend cells in cold PBS. Make aliquots of 4 million cells per 15 ml conical tube. Label tubes correctly. Spin down again and aspirate supernatant. Snap freeze in liquid nitrogen or a dry ice/100% ethanol bath, and store at -80 °C.

Day 3

  1. Remove 2 untreated and 2 treated tubes from the -80 °C freezer. Place on ice to thaw.
    Note: All tubes on ice from now on. 
  2. Resuspend cell pellet in 200 μl cold EBC buffer with protease inhibitors (complete mini EDTA-free tablet, Roche. I make up 10 ml of EBC buffer with 1 tablet of the complete mini EDTA-free protease inhibitor cocktail). One set of untreated and treated will be used for IP for p65 and the other will be a minus antibody (-Ab) control. So you will have 4 tubes total and will need 800 μl cold EBC buffer+protease inhibitors.
  3. Sonicate samples at 12% output, 10 sec x 5 cycles, with 30 sec on ice between sonications.
  4. Spin cells down at 9,000 x g for 10 min.
  5. Remove supernatant into a fresh tube.
  6. Follow the protocol for the Chromatin Immunoprecipitation Assay Kit.
    1. Make a master mix of protease inhibitor cocktail solution with ChIP dilution buffer (included in the kit). Since there are 4 total samples, you will need 6.4 ml ChIP dilution buffer and 800 μl of the 10x protease inhibitor cocktail solution. Add 1,800 μl to each sample. Remove 20 μl from the untreated and treated –Ab tubes as input controls and save at 4 °C for later use.
    2. Preclear the lysates with 75 μl Protein A/G PLUS-agarose for 1 h rotating at 4 °C.
    3. Pellet at 14,000 x g for ~15 sec. Save the supernatant to a fresh tube, being careful to not disturb the pellet. I usually leave about 50 μl of supernatant behind so as not contaminate the supernatant with the agarose. Keep the tubes with the supernatant, toss the pellets in the trash.
    4. For the –Ab tubes, rotate at 4 °C overnight. For the p65 IPs, use 2 μg of p65 antibody per tube. Rotate overnight at 4 °C.

Day 4

  1. Add 60 μl of Protein A/G PLUS-agarose to all 4 tubes (–Ab tubes. Rotate for 1 h at 4 °C to pull down immuno complexes.
  2. Briefly centrifuge pellets at 14,000 x g, 15 sec. Remove supernatant and discard.
  3. Wash pellet with the following list of buffers that are included in the kit. If you do not have the kit, the recipe is included. Centrifuge after washing, remove supernatant completely and resuspend in the next buffer on this list. The wash buffers increase in salt concentration to remove non-specific binding to the antibody/agarose complex. Use 500 μl of the buffer, rotating for 5 min at 4 °C for each wash.
    1. 1x low salt immune complex wash buffer.
    2. 1x high salt immune complex wash buffer.
    3. 1x LiCl immune complex wash buffer.
    4. 2x TE buffer.
  4. Freshly prepare elution buffer. Elute protein by adding 250 μl of elution buffer, vortexing, and incubating at RT for 15 min with rotation. Briefly spin, remove eluate to a new tube, and repeat elution with another 250 μl elution buffer. Combine eluents for a final volume of 500 μl per IP reaction (at this point you should have 4 tubes, 2 for p65 IP, 2 for -Ab, with control and treated for each).
  5. Add 20 μl 5 M NaCl to the eluents. Also, add 5 μl 5 M NaCl to the input controls and heat all samples at 65 °C for 4 h to reverse the crosslinking. At this step the samples can be stored at –20 °C, or proceed to DNA extraction if you have time.

Day 5

  1. Recover DNA by phenol/chloroform extraction.
    1. Measure the volume of the reversed-cross linked samples. Add an equal volume of phenol to each sample, vortex, and spin for 3 min at 14,000 x g. Transfer supernatant to a fresh tube (avoid the bottom and interphase layer).
    2. Add an equal volume of chloroform, vortex, and spin for 3 min at 14,000 x g. Transfer supernatant to a fresh tube (avoid the bottom and interphase layer).
  2. Precipitate DNA by ethanol precipitation.
    1. Measure the volume of the extracted samples. Add 0.1 volume 3 M sodium acetate, 2 volumes of 100% ethanol, and 4 μl of 5 mg/ml glycogen (as a DNA carrier) to each tube.
    2. Vortex and place at -20 °C for 30 min.
    3. Spin for 20 min at 14,000 x g at 4 °C.
    4. Very carefully pour off the supernatant, watching the DNA pellet very carefully so that it does not dislodge.
    5. Wash pellets with 500 μl 70% ethanol. Vortex and spin for 5 min at 14,000 x g at 4 °C.
    6. Very carefully pour off the supernatant, watching the DNA pellet so that it does not dislodge. It will be very “slippery” at this point. I use an unfiltered tip to wick away the remaining ethanol from the pellet and to push the pellet back to the bottom of the tube if it has moved.
    7. Flip the tube upside-down and air dry for 7 min.
    8. Resuspend in 30 μl water.
  3. Proceed with PCR using primers surrounding the potential binding site of interest in the gene promoter. Product size of DNA should be around 100-200 bp. Run PCR reaction on a 2% TBE or TAE gel and document.

Recipes

  1. EBC buffer
    50 mM Tris (pH 7.5)
    100 mM NaCl
    0.5% NP-40
  2. ChIP dilution buffer
    0.01% SDS
    1.1% Triton X-100
    1.2 mM EDTA
    16.7 mM Tris-HCl (pH 8.1)
    167 mM NaCl
  3. 10x protease inhibitor solution
    Dissolve one complete mini EDTA-free tablet in 1 ml water.
  4. Low salt immune complex wash buffer
    0.1% SDS
    1% Triton X-100
    2 mM EDTA
    20 mM Tris-HCl (pH 8.1)
    150 mM NaCl
  5. High salt immune complex wash buffer
    0.1% SDS
    1% Triton X-100
    2 mM EDTA
    20 mM Tris-HCl (pH 8.1)
    500 mM NaCl
  6. LiCl immune complex wash buffer
    0.25 M LiCl
    1% IGEPAL-CA630
    1% deoxycholic acid (sodium salt)
    1 mM EDTA
    10 mM Tris (pH 8.1)
  7. TE buffer
    10 mM Tris-HCl
    1 mM EDTA (pH 8.0)
  8. Elution buffer
    1% SDS
    0.1 M NaHCO3

Acknowledgments

This protocol was adapted from Dudgeon et al. (2012). This work was supported by NIH grants CA106348 and CA121105 and American Cancer Society grant RSG-07-156-01-CNE (LZ); Flight Attendant Medical Research Institute, NIH grant CA129829 and American Cancer Society grant RGS-10-124-01-CCE (JY); NIH National Research Service Award postdoctoral fellowship grant F32CA139882 (CD); and China Scholarship Council (RP). LZ is a scholar of the V Foundation for Cancer Research.

References

  1. Dudgeon, C., Peng, R., Wang, P., Sebastiani, A., Yu, J. and Zhang, L. (2012). Inhibiting oncogenic signaling by sorafenib activates PUMA via GSK3β and NF-κB to suppress tumor cell growth. Oncogene 31(46): 4848-4858.
  2. Wang, P., Yu, J. and Zhang, L. (2007). The nuclear function of p53 is required for PUMA-mediated apoptosis induced by DNA damageProc Natl Acad Sci U S A 104(10): 4054-4059.

简介

Chromatin Immunoprecipitation (ChIP) is an important procedure that allows you to verify if a certain protein is physically located at a regulatory region. This information, taken together with other procedures such as luciferase assays and EMSAs, will give definitive proof that the query protein is involved in the transcription of a protein. This procedure for p65 ChIP can be adapted to investigate other proteins; just a change of the antibody will suffice.
The transcription factor known as NF-κB is a homo- or hetero-dimer consisting of members of the Rel/NFKB family. The most abundant NF-κB complexes are made of two different proteins, p65 (Rel-A) and p50 (NFKB1). The NF-κB complex is initially inhibited by IκB by direct binding, thus trapping NF-κB in the cytoplasm. After a stimulatory signal, IκB kinase (IKK) phosphorylates IκB, allowing IκB to undergo proteasome-mediated degradation. The degradation of IκB and phosphorylation of p65 by multiple kinases activates NF-κB, allowing it to transport to the nucleus and cause the transcriptional activation of many of its target genes containing κB sites (consensus sequence: gggRNNYYcc, R = purine Y = pyrimidine), such as PUMA, IL-6, and TNF.

关键字:P65, NFκ, 炸薯条, 发起人

材料和试剂

  1. HCT116细胞系
  2. 胰蛋白酶(0.05%)(Life Technologies,目录号:25300-054)
  3. DMSO
  4. PBS
  5. 甲醛(J.T.Baker,目录号:2106-01)
  6. 1 M甘氨酸
  7. 液氮或干冰/100%乙醇
  8. NP-40
  9. 蛋白酶抑制剂混合物片剂(F.Hoffmann-La Roche,目录号:04-693159-001)
  10. p65抗体(Santa Cruz,目录号:sc-109)
  11. 染色质免疫沉淀测定试剂盒(EMD Millipore,目录号:17-295)在线: http://www.millipore.com/coa.nsf/a73664f9f981af8c852569b9005b4eee/20c0dc520d2b30f0852573be007ffdae/$FILE/17-295-DAM1411265.pdf
  12. 蛋白A/G PLUS-琼脂糖(已经用BSA预封闭)(Santa Cruz,目录号:sc-2003)
  13. 苯酚
  14. 氯仿
  15. 糖原
  16. 70%乙醇
  17. SDS
  18. Triton X-100
  19. EDTA
  20. Tris-HCl
  21. NaCl
  22. NaHCO 3
  23. EBC缓冲区(参见配方)
  24. ChIP稀释缓冲液(参见配方)
  25. 10x蛋白酶抑制剂溶液(参见配方)
  26. 低盐免疫复合物洗涤缓冲液(见配方)
  27. 高盐免疫复合物洗涤缓冲液(参见配方)
  28. LiCl免疫复合物洗涤缓冲液(参见配方)
  29. TE缓冲区(参见配方)
  30. 洗脱缓冲液(见配方)

设备

  1. 超声波仪(Branson数字超声波仪450)(Branson Ultrasonics公司)
  2. 离心机
  3. 15ml锥形管
  4. T75瓶
  5. 细胞刮刀

程序

大纲

  1. 开始这个程序前三天,通常在星期五,分裂细胞1:4,使他们将在星期一汇合。
  2. 第1天(星期一) - 分隔您的单元格。
  3. 天   2(星期二) - 你的细胞与你发现的任何化合物   导致感兴趣的基因的转录激活。 修复单元格 当你相信p65会在你的基因的调节区 利益。 计数细胞并等分4百万个细胞每15ml锥形 管
  4. 第3天(星期三)用p65抗体过夜对样品,预清除样品和IP进行超声处理
  5. 第4天(星期四) - 洗涤,洗脱和反向交联。
  6. 天   5(星期五) - 苯酚/氯仿萃取和乙醇沉淀。 如果你有一天的时间,继续做感兴趣的区域的PCR。

注意:首先确定您的特定细胞类型的超声处理条件,以产生300-1,000bp之间的DNA片段。 这通常是大约12%的输出,5个循环,每个10秒,在声处理之间具有30秒的冷却期。 如图1的第二泳道所示,DNA的1.2%琼脂糖溴化乙锭凝胶在该区域将显示更明显的污迹。



图1.确定HCT116对ChIP的超声处理条件 用星号表示的泳道显示了正确的超声处理条件。

第1天

拆分您的单元格。细胞应处于对数生长期。通常我使用T75烧瓶培养细胞。第二天细胞应该约60-70%汇合。如果你用3ml胰蛋白酶+ 9ml培养基分离一个融合的T75细胞瓶,你将需要4ml细胞(〜5×10 6个细胞)每个T75(你可以做3总,因此未处理,处理和每个T75烧瓶的计数皿)。这些条件适用于HCT116,因此应根据您的细胞系进行相应调整。

第2天

  1. 第二天用DMSO或PBS处理细胞,用于对照(取决于您的化合物的溶解度)和目标化合物。为了在索拉非尼处理后检测PUMA启动子上的p65,我进行了8小时时间点。 这可能会根据蛋白质的转录速率而改变。我会用你第一次开始看到你的蛋白质表达增加的时间
  2. 当你等待你的时间点,计数你的额外烧瓶中的细胞。这将告诉你在你的其他烧瓶中有多少细胞/ml。如果你不能这样做,由于有限的样品量,在你的化合物的时间点后,在修复细胞之前,计数它们。
  3. 在烧瓶中加入甲醛至终浓度为1%。让我们坐在室温(RT)与温柔摇动15分钟。
  4. 加入1M甘氨酸至终浓度为125mM以终止反应。轻轻混匀。
  5. 使用细胞刮刀,刮去细胞并在4℃下以400×g离心5分钟使细胞离心。吸出上清液。
  6. 重悬细胞在冷PBS。每15毫升锥形管制备400万细胞的等分试样。标签管正确。再次旋转并吸出上清液。在液氮或干冰/100%乙醇浴中快速冷冻,并储存在-80℃

第3天

  1. 从-80℃冰箱中取出2个未处理和2个处理的管。 放在冰上解冻。
    注意:从现在开始,所有管都在冰面上。 
  2. 将细胞沉淀重悬于200μl具有蛋白酶抑制剂的冷EBC缓冲液(完全无EDTA的片剂,Roche.I组成10ml EBC缓冲液,1片完全无EDTA的蛋白酶抑制剂混合物)。 一组未处理和处理的将用于p65的p65,而另一组将是负抗体(-Ab)对照。 所以你将有4支管,将需要800μl冷EBC缓冲液+蛋白酶抑制剂。
  3. 超声处理样品在12%输出,10秒×5个循环,在声处理之间在冰上30秒。
  4. 将细胞以9,000×g离心10分钟。
  5. 将上清液移至新管中。
  6. 按照协议的染色质免疫沉淀测定试剂盒。
    1. 使用ChIP稀释缓冲液(包含在试剂盒中)制备蛋白酶抑制剂混合物溶液的主混合物。由于总共有4个样品,您将需要6.4 ml ChIP稀释缓冲液和800μl的10x蛋白酶抑制剂混合液。每个样品加入1,800μl。从未处理和处理的-Ab管作为输入控件取出20微升,保存在4℃供以后使用
    2. 用75μl蛋白A/G PLUS-琼脂糖在4℃下旋转1小时来预清洗裂解物。
    3. 粒料在14,000×g 约15秒。保存上清液到一个新的管,小心不要打扰沉淀。我通常留下约50微升的上清液后面,以免污染上清液与琼脂糖。保持管与上清液,将球团扔在垃圾桶。
    4. 对于-Ab管,在4℃下旋转过夜。 对于p65 IP,每管使用2μg的p65抗体。 在4℃下旋转过夜。

第4天

  1. 添加60微升蛋白A/G PLUS琼脂糖到所有4管(-Ab管。在4℃下旋转1小时,以拉下免疫复合物。
  2. 以14000×g /秒,15秒钟短暂离心沉淀。 除去上清液并丢弃。
  3. 使用以下试剂盒中包含的缓冲液列表清洗沉淀。 如果您没有套件,则包括配方。 离心后洗涤,完全去除上清液,并在此列表中的下一个缓冲液中重悬。 洗涤缓冲液的盐浓度增加以去除与抗体/琼脂糖复合物的非特异性结合。 使用500μl的缓冲液,在4℃下旋转5分钟,每次洗涤。
    1. 1x低盐免疫复合物洗涤缓冲液
    2. 1×高盐免疫复合物洗涤缓冲液
    3. 1×LiCl免疫复合物洗涤缓冲液
    4. 2x TE缓冲区。
  4. 新制备洗脱缓冲液。 通过加入250μl洗脱缓冲液洗脱蛋白质,涡旋,并在室温下旋转孵育15分钟。 短暂旋转,将洗脱液移至新管中,并用另一250μl洗脱缓冲液重复洗脱。 合并洗脱液,每次IP反应的最终体积为500μl(此时,您应该有4个试管,2个用于p65 IP,2个用于-Ab,有控制并分别处理)。
  5. 向洗脱液中加入20μl5 M NaCl。 另外,向输入对照中加入5μl5M NaCl,并在65℃加热所有样品4小时以逆转交联。 在这一步,样品可以储存在-20°C,或如果你有时间,进行DNA提取。

第5天

  1. 通过苯酚/氯仿萃取回收DNA。
    1. 测量反向交联样品的体积。 向每个样品中加入等体积的苯酚,涡旋,并以14,000×g旋转3分钟。 将上清转移到新管(避免底部和中间相层)。
    2. 加入等体积的氯仿,涡旋,并以14,000×g旋转3分钟。 将上清转移到新管(避免底部和中间相层)。
  2. 通过乙醇沉淀沉淀DNA。
    1. 测量提取样品的体积。 向每个管中加入0.1体积3M乙酸钠,2体积100%乙醇和4μl5mg/ml糖原(作为DNA载体)。
    2. 涡旋并置于-20℃下30分钟
    3. 在4℃以14,000×g离心20分钟。
    4. 非常小心地倒出上清液,仔细观察DNA沉淀,使其不会脱落。
    5. 用500μl70%乙醇洗涤沉淀。 涡旋并在4℃以14,000×g离心5分钟
    6. 非常小心地倒出上清液,观察DNA沉淀,使其不会脱落。 这将是非常"滑"。 我使用未过滤的吸头从芯块中取出剩余的乙醇,如果芯块移动,则将芯块推回到管的底部。
    7. 将管翻转倒置并风干7分钟。
    8. 重悬在30μl水中。
  3. 使用围绕基因启动子中感兴趣的潜在结合位点的引物进行PCR。 DNA的产物大小应为约100-200bp。 在2%TBE或TAE凝胶和文件上进行PCR反应。

食谱

  1. EBC缓冲区
    50mM Tris(pH7.5) 100 mM NaCl
    0.5%NP-40
  2. ChIP稀释缓冲液
    0.01%SDS
    1.1%Triton X-100 1.2mM EDTA
    16.7mM Tris-HCl(pH8.1) 167 mM NaCl
  3. 10x蛋白酶抑制剂溶液
    将一个完整的迷你无EDTA片溶于1ml水中
  4. 低盐免疫复合物洗涤缓冲液
    0.1%SDS
    1%Triton X-100 2mM EDTA 20mM Tris-HCl(pH8.1) 150mM NaCl
  5. 高盐免疫复合物洗涤缓冲液
    0.1%SDS
    1%Triton X-100 2mM EDTA 20mM Tris-HCl(pH8.1) 500 mM NaCl
  6. LiCl免疫复合物洗涤缓冲液
    0.25 M LiCl
    1%IGEPAL-CA630
    1%脱氧胆酸(钠盐)
    1mM EDTA
    10mM Tris(pH8.1)
  7. TE缓冲区
    10mM Tris-HCl
    1mM EDTA(pH8.0)
  8. 洗脱缓冲液
    1%SDS
    0.1 M NaHCO 3 3/v/v

致谢

该协议改编自Dudgeon等人(2012)。这项工作由NIH资助CA106348和CA121105和美国癌症协会授权RSG-07-156-01-CNE(LZ)支持;飞行员医学研究所,NIH资助CA129829和美国癌症协会授予RGS-10-124-01-CCE(JY); NIH国家研究服务奖博士后奖学金F32CA139882(CD);和中国奖学金委员会(RP)。 LZ是V基金会癌症研究的学者。

参考文献

  1. Dudgeon,C.,Peng,R.,Wang,P.,Sebastiani,A.,Yu,J.and Zhang,L。 p53的核功能是由DNA损伤诱导的PUMA介导的凋亡所必需的。< em> Proc Natl Acad Sci > USA 104(10):4054-4059。
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引用:Dudgeon, C. R. (2013). p65 Chromatin Immunoprecipitation Protocol. Bio-protocol 3(8): e683. DOI: 10.21769/BioProtoc.683.
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