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Estradiol Receptor (ER) Chromatin Immunoprecipitation in MCF-7 Cells
MCF-7 细胞中的雌激素受体(ER)染色质免疫共沉淀   

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Abstract

Steroid hormone receptors, for example estradiol receptor, act like transcription factors. In the cell, steroids bind to a specific receptor. Upon ligand binding, many steroid receptors dimerize and enter nuclei where they bind specific DNA sequences called Hormone Responsive Elements (HRE) and regulate gene transcription. ER is able to bind DNA sites that are not Estrogen Responsive Elements (ERE) so regulating also the transcription of genes that are not classically controlled by estrogens.

Keywords: Estradiol Receptor(雌二醇受体), Breast cancer(乳腺癌), ChIP(炸薯条), MCF-7 cells(MCF-7细胞), Transcription factor(转录因子)

Materials and Reagents

  1. Fetal Bovine Serum (FBS) (Life Technologies, Gibco®, catalog number: 10270 )
  2. 200 mM L-Glutamine (100x) (Life Technologies, Gibco®, catalog number: 25030 )
  3. Penicillin-Streptomycin (100x) (Life Technologies, Gibco®, catalog number: 15140-122 )
  4. Hydrocortisone (Sigma-Aldrich, catalog number: H-0888 )
  5. Insulin (Human, recombinant) (F. Hoffmann-La Roche, catalog number: 11376497001 )
  6. Estradiol (Beta-estradiol) (Sigma-Aldrich, catalog number: E8875 )
  7. Protease inhibitors cocktail tablets (LAP tablets) (F. Hoffmann-La Roche, catalog number: 11836153001 )
  8. Protein A-agarose fast flow (Sigma-Aldrich, catalog number: P3476 )
  9. Formaldeyde (Sigma-Aldrich)
  10. Salmon sperm DNA
  11. DPBS (Sigma-Aldrich, catalog number: D5652 )
  12. Proteinase K (F. Hoffmann-La Roche, catalog number: 0 3115879001 )
  13. Qiagen PCR purification kit
  14. Immunoprecipitating antibody (ER-alpha) (Sigma-Aldrich, catalog number: E1396 )
  15. Primers CCDN1 locus (-1039 to 770 bp): Fw(-1039) (AACAAAACCAATTAGGAACCTT), Rv(?770) (ATTTCCTTCATCTTGTCCTTCT)
  16. Primers CCDN1 promoter (-235 to -53 bp): Fw(-235) (TATGAAAACCGGACTACAGG), and Rv(?53) (CTGTTGTTAAGCAAAGATCAAAG)
  17. Charcoal dextran stripped serum (CSS) (see Recipes)
  18. Phenol Red-free Dulbecco’s Modified Medium (DMEM with PR) (Life Technologies, Gibco®, catalog number: 31885 ) (for MCF-7 cells) (see Recipes)
  19. Phenol Red-free Dulbecco’s Modified Medium (DMEM w/o PR) (Life Technologies, Gibco®, catalog number: 11880 ) supplemented with Charcoal (for MCF-7 cells) (see Recipes)
  20. 50x LAP (Protease inhibitor cocktail) (see Recipes)
  21. DTT solution (see Recipes)
  22. Buffer I (see Recipes)
  23. Buffer II (see Recipes)
  24. Buffer III or cell lysis buffer (see Recipes)
  25. Buffer IV (see Recipes)
  26. TSE I buffer (see Recipes)
  27. TSE II buffer (see Recipes)
  28. TSE III buffer (see Recipes)
  29. TE buffer (see Recipes)
  30. Elution buffer (see Recipes)
  31. PCR buffer (see Recipes)

Equipment

  1. Sonicator (equipped with a 3 mm diameter tip) (Sonics & Materials Inc., model: Vibracell VC 130PB )
  2. 100 mm dishes
  3. Centrifuge (Eppendorf centrifuge, model: 5417R )
  4. Shaker
  5. PCR thermal cycler

Procedure

  1. The MCF-7 cells are plated in 100 mm dishes approximately at 40-50% cell confluence in Phenol red Dulbecco’s modified medium. After12-18 h, the Red phenol medium is substituted with Phenol red-free Dulbecco’s modified medium supplemented with CSS. Cells are maintained in this medium for 3 or 4 days (80% confluence in 100 mm dishes).
  2. Stimulate the cells with 10 nM estradiol for various times from 15 to 75 min. (the maximal ER binding to chromatin is usually observed after 30-40 min of hormone treatment). Use un-stimulated cells as negative control.
  3. After hormone stimulation, wash the cells twice with cold PBS (5-10 ml).
  4. Cross-link the cells with 10 ml 1% formaldehyde solution in PBS at room temperature for 10 min. Add the formaldehyde to PBS immediately prior to use.
  5. Rinse cells twice with cold PBS (5-10 ml).
  6. Add 1 ml DTT solution and collect the cells in a 1.5 ml tube using a cell scraper.
  7. After collecting, incubate the cells for 15 min at 30 °C and centrifuge for 4 or 5 min at 3,000 RCF.
  8. Wash the pellet three times with 1 ml PBS and after every wash centrifuge at 3,000 RCF for 3 min.
  9. Wash sequentially the pellet with 1 ml Buffer I with 50x LAP, 1 ml Buffer II with LAP.
  10. Suspend the cellular pellet in 300 μl Buffer III with 50x LAP and incubate 10 min on ice.
  11. Sonicate three times at 30-40% amplitude and 0.4 W intensity for 35 sec each to obtain 200 bp DNA fragments. During the sonication, keep the samples on ice.
  12. To define amplitude, intensity and duration of sonication, you can use a sample of MCF-7 cells, sonicate at different intensity, amplitude and duration and after check the DNA fragment size running the sonicated DNA on an agarose gel. The right conditions will be those that will allow you to obtain a DNA smear with the center on around 200 bp.
  13. Centrifuge the cellular lysates at 20,000 RCF for 10 min at 4 °C, collect the supernatants and dilute ten fold using the Buffer IV with 50x LAP. Store in a rack and keep at 4 °C for 3 or 4 h or overnight (O.N.).
  14. For preparing the protein A-agarose (40 μl of 50% for each sample), wash twice the required amount of resin with TE solution (ten fold the protein A-agarose volume). Collect the protein A-agarose by brief centrifugation. After the last wash, discard the TE solution and add fresh TE solution and salmon sperm DNA (20 μl TE solution and 2 μg salmon sperm DNA for each sample). Keep on a rotating platform at 4 °C for 3 or 4 h or O.N.
  15. Clear the samples at 20,000 RCF for 10 sec. Store at -80 °C or use immediately for immunoprecipitation and lysates. Save back 20% of the total supernatant as total input control and process with eluted IPs beginning with the cross-link reversal step.
  16. Add the immunoprecipitating antibody (2 μl ER-alpha) to the 0.7 ml samples fraction in a new tube and precipitate for 6 h or overnight. For a negative control, incubate 0.7 ml samples fraction with 1 μl (2 μg) of rabbit IgG. Keep all the samples on a rotating platform at 4 °C.
  17. Add 40 μl of blocked protein A-agarose beads at 4 °C with rotation O.N. if you have incubated the samples with the antibody for 6 h or for 2-3 h if you have incubated the samples with the antibody O.N.
  18. Pellet beads by centrifugation (3,000 x g) at 4 °C and wash for 10 min sequentially with 1 ml of TSE I, 1 ml of TSE II and 500 μl of TSE III. Wash three times with 1 ml TE Buffer.
  19. Elute with 300 μl of freshly prepared elution buffer. Prepare buffer fresh each time. Vortex briefly to mix and shake gently on the vortex shaker for 10 min.
  20. Centrifuge at 20,000 rpm for 10 min, transfer supernatants to clean tube and add 200 μl elution buffer in 100 μl of the frozen input control.
  21. Reverse formaldehyde cross-linking by heating at 65 °C O.N.
  22. Add 18 μl 1 M Tris-HCl pH 6.5 0.5 M EDTA and 5 μg Proteinase K to each sample and heat at 48 °C for 90 min.
  23. Isolate DNA by using either the Qiagen PCR purification kit.
  24. Another method for isolating DNA is the 1x phenol/chloroform extraction, 1x chloroform extraction, O.N. precipitation using 2.5 volumes absolute ethanol with 30 μl potassium acetate 3 M pH 5.7 and 5 μg glycogen. Wash pellets twice with 500 μl 70% ethanol and air dried.
  25. Resuspend pellets in 35 μl TE with 0.1 mg/ml RNase A.
  26. Perform the conventional PCR using 2 μl DNA per reaction. For example, for CCDN1 locus (-1039 to -770 bp) or for CCDN1 promoter (-235 to -53 bp) PCR, use 20 μl H2O solution with 1.5 mM MgCl2, 0.2 mM dNTP, 0.25 μM PRIMER F, 0.25 μM PRIMER R, 0.5 U/μl TAQ Pol, 2 μl DNA and 2 μl PCR buffer. Use 33-35 cycles of amplification (denaturation 96 °C for 20 sec, annealing 50 °C for 90 sec, elongation 69 °C for 60 sec). Run in a 2% agarose gel in TBE 0.5%.

Recipes

  1. Charcoal dextran stripped serum
    Dextran coated charcoal is used to strip steroid hormones from serum. Charcoal/dextran stripped serum is commercially available, but we prepare the serum as described in Migliaccio et al. (2011).
  2. Phenol red-free Dulbecco’s modified medium for MCF-7 cells (DMEM with PR)
    5% FBS
    2 mM L-glutamine
    100 U/ml penicillin-streptomycin
    3.75 ng/ml Hydrocortisone
    6 ng/ml Insulin
  3. Phenol red-free Dulbecco’s modified medium supplemented with Charcoal for MCF-7 cells
    (DMEM w/o PR)
    5% CSS
    2 mM L-glutamine
    100 U/ml penicillin-streptomycin
    3.75 ng/ml Hydrocortisone
    6 ng/ml Insulin
  4. 50x LAP (Protease inhibitor cocktail)
    Dissolve a tablet in 1 ml distilled H2O and use 50x
  5. DTT solution
    10 mM DTT
    100 mM Tris-HCl
    pH 9.5
  6. Buffer I
    0.25% Triton X-100
    10 mM EDTA
    0.5 mM EGTA
    10 mM Hepes
    pH 6.5
  7. Buffer II
    200 mM NaCl
    1 mM EDTA
    0.5 mM EGTA
    10 mM Hepes
    pH 6.5
  8. Buffer III or cell lysis buffer
    1% SDS
    10 mM EDTA
    50 mM Tris-HCl (pH 8.1)
    50x protease inhibitor cocktail
  9. Buffer IV
    1% Triton X-100
    2 mM EDTA
    150 mM NaCl
    20 mM Tris-HCl (pH 8.1)
  10. TSE I buffer
    0.1% SDS
    1% Triton X-100
    2 mM EDTA
    20 mM Tris-HCl (pH 8.1)
    150 mM NaCl
  11. TSE II buffer
    0.1% SDS
    1% Triton X-100
    2 mM EDTA
    20 mM Tris-HCl (pH 8.1)
    500 mM NaCl
  12. TSE III buffer
    0.25 M LiCl
    1% NP40
    1% Sodium Deoxycholate
    1 mM EDTA
    10 mM Tris-HCl (pH 8.1)
  13. TE buffer
    20 mM Tris-HCl (pH 8.1)
    1 mM EDTA (pH 8.0)
  14. Elution buffer
    1% SDS
    0.1 M NaHCO3
  15. PCR buffer
    300 mM Tris-Base
    100 mM Hepes
    250 mM Potassium Chloride
    200 mM Potassium Glutamate
    20 mM DTT
    50% Glycerol
    Sterilize by filtration

Acknowledgments

This work was funded by the Italian Association for Cancer Research (A.I.R.C.; Grant No. IG 5389). Pia Giovannelli is supported by a fellowship from A.I.R.C. This protocol was adapted from Castoria et al. (2012).

References

  1. Castoria, G., Giovannelli, P., Lombardi, M., De Rosa, C., Giraldi, T., de Falco, A., Barone, M. V., Abbondanza, C., Migliaccio, A. and Auricchio, F. (2012). Tyrosine phosphorylation of estradiol receptor by Src regulates its hormone-dependent nuclear export and cell cycle progression in breast cancer cells. Oncogene 31(46): 4868-4877.
  2. Lombardi, M., Castoria, G., Migliaccio, A., Barone, M. V., Di Stasio, R., Ciociola, A., Bottero, D., Yamaguchi, H., Appella, E. and Auricchio, F. (2008). Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells. J Cell Biol 182(2): 327-340. 
  3. Migliaccio, A., Castoria, G., Auricchio, F. (2011). Analysis of androgen receptor rapid actions in cellular signaling pathways: receptor/Src association. Methods Mol Biol 776: 361-370.
  4. Yahata, T., Shao, W., Endoh, H., Hur, J., Coser, K. R., Sun, H., Ueda, Y., Kato, S., Isselbacher, K. J., Brown, M. and Shioda, T. (2001). Selective coactivation of estrogen-dependent transcription by CITED1 CBP/p300-binding protein. Genes Dev 15(19): 2598-2612.

简介

类固醇激素受体,例如雌二醇受体,像转录因子一样起作用。 在细胞中,类固醇与特异性受体结合。 在配体结合时,许多类固醇受体二聚化并进入细胞核,其中它们结合称为激素应答元件(HRE)的特异性DNA序列并调节基因转录。 ER能够结合不是雌激素反应元件(ERE)的DNA位点,从而也调节不经典地由雌激素控制的基因的转录。

关键字:雌二醇受体, 乳腺癌, 炸薯条, MCF-7细胞, 转录因子

材料和试剂

  1. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10270)
  2. 200mM L-谷氨酰胺(100x)(Life Technologies,Gibco ,目录号:25030)
  3. 青霉素 - 链霉素(100x)(Life Technologies,Gibco ,目录号:15140-122)
  4. 氢化可的松(Sigma-Aldrich,目录号:H-0888)
  5. 胰岛素(人,重组)(F.Hoffmann-La Roche,目录号:11376497001)
  6. 雌二醇(β-雌二醇)(Sigma-Aldrich,目录号:E8875)
  7. 蛋白酶抑制剂混合片剂(LAP片剂)(F.Hoffmann-La Roche,目录号:11836153001)
  8. 蛋白A-琼脂糖快速流(Sigma-Aldrich,目录号:P3476)
  9. Formaldeyde(Sigma-Aldrich)
  10. 鲑鱼精子DNA
  11. DPBS(Sigma-Aldrich,目录号:D5652)
  12. 蛋白酶K(F.Hoffmann-La Roche,目录号:03115879001)
  13. Qiagen PCR纯化试剂盒
  14. 免疫沉淀抗体(ER-α)(Sigma-Aldrich,目录号:E1396)
  15. 引物CCDN1基因座(-1039至770bp):Fw(-1039)(AACAAAACCAATTAGGAACCTT),Rv(λ770)(ATTTCCTTCATCTTGTCCTTCT)
  16. 引物CCDN1启动子(-235至-53bp):Fw(-235)(TATGAAAACCGGACTACAGG)和Rv(λ53)(CTGTTGTTAAGCAAAGATCAAAG)
  17. 木炭葡聚糖剥离血清(CSS)(参见配方)
  18. 酚红无Dulbecco改良培养基(具有PR的DMEM)(Life Technologies,Gibco ,目录号:31885)(用于MCF-7细胞)(参见Recipes)
  19. 补充有炭(对于MCF-7细胞)(参见Recipes)的无酚红的Dulbecco's改良培养基(DMEM w/o PR)(Life Technologies,Gibco ,目录号:11880) >
  20. 50x LAP(蛋白酶抑制剂混合物)(参见配方)
  21. DTT解决方案(参见配方)
  22. 缓冲区I(参见配方)
  23. 缓冲液II(参见配方)
  24. 缓冲液III或细胞裂解缓冲液(参见配方)
  25. 缓冲液IV(参见配方)
  26. TSE I缓冲区(请参阅配方)
  27. TSE II缓冲区(参见配方)
  28. TSE III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)
  31. PCR缓冲液(见配方)

设备

  1. 超声波仪(配备有3mm直径的尖端)(Sonics& Materials Inc.,型号:Vibracell VC 130PB)
  2. 100毫米培养皿
  3. 离心机(Eppendorf离心机,型号:5417R)
  4. 振动器
  5. PCR热循环仪

程序

  1. 将MCF-7细胞接种在100mm皿中,在苯酚红Dulbecco改良培养基中约40-50%细胞汇合。 12-18小时后,用补充有CSS的无酚红的Dulbecco's改良培养基代替红色酚培养基。 将细胞维持在该培养基中3或4天(在100mm培养皿中80%汇合)。
  2. 刺激细胞与10 nM雌二醇不同时间从15至75分钟。 (通常在激素治疗30-40分钟后观察到最大ER结合至染色质)。 使用未刺激细胞作为阴性对照。
  3. 激素刺激后,用冷PBS(5-10ml)洗涤细胞两次
  4. 交联细胞与10ml 1%甲醛的PBS溶液在室温下10分钟。 在使用前立即将甲醛加入PBS中。
  5. 用冷PBS(5-10ml)冲洗细胞两次
  6. 加入1ml DTT溶液,并使用细胞刮刀将细胞收集在1.5ml管中
  7. 收集后,在30℃下孵育细胞15分钟,并在3,000RCF离心4或5分钟。
  8. 用1ml PBS洗涤沉淀物3次,每次洗涤后,以3000RCF离心3分钟
  9. 用1ml缓冲液I用50x LAP,1ml缓冲液II用LAP顺序洗涤沉淀
  10. 悬浮细胞沉淀在300微升缓冲液III与50x LAP和在冰上孵育10分钟
  11. 在30-40%振幅和0.4W强度下超声处理三次,每次35秒,以获得200bp的DNA片段。在超声处理期间,将样品保持在冰上
  12. 要定义超声处理的幅度,强度和持续时间,您可以使用MCF-7细胞样品,在不同强度,幅度和持续时间超声,并检查在琼脂糖凝胶上运行超声处理的DNA的DNA片段大小。正确的条件将是允许您获得中心在大约200 bp的DNA涂片的条件
  13. 离心细胞裂解液在20,000 RCF在4°C 10分钟,收集上清液,稀释10倍,使用缓冲液IV与50x LAP。存放在架子上,并在4°C保持3或4小时或过夜(O.N.)
  14. 为了制备蛋白A-琼脂糖(每个样品40μl50%),用TE溶液洗涤所需量的树脂两次(蛋白A-琼脂糖体积的10倍)。通过短暂离心收集蛋白A-琼脂糖。最后一次洗涤后,丢弃TE溶液,并加入新鲜的TE溶液和鲑鱼精子DNA(每个样品20μlTE溶液和2μg鲑鱼精子DNA)。保持在4°C的旋转平台3或4小时或O.N。
  15. 在20,000RCF下清除样品10秒。储存于-80°C或立即使用免疫沉淀和裂解物。将总上清液的20%保存为总输入对照,并以交联反转步骤开始的洗脱IP进行处理。
  16. 将免疫沉淀抗体(2μlER-α)加入新管中的0.7ml样品级分中,沉淀6小时或过夜。对于阴性对照,用1μl(2μg)兔IgG孵育0.7ml样品级分。将所有样品保存在4℃的旋转平台上。
  17. 在4℃下加入40μl封闭的蛋白A-琼脂糖珠,旋转O.N.如果你用抗体孵育样品6小时或2-3小时,如果你用抗体O.N.孵育样品。
  18. 通过在4℃下离心(3,000xg)沉淀小球珠,并用1ml TSE I,1ml TSE II和500μlTSE III依次洗涤10分钟。用1ml TE缓冲液洗涤三次。
  19. 用300μl新鲜制备的洗脱缓冲液洗脱。每次准备缓冲液新鲜。短暂涡旋混合并在涡旋振荡器上轻轻摇动10分钟。
  20. 以20,000rpm离心10分钟,将上清液转移至清洁管,并在100μl冷冻输入对照中加入200μl洗脱缓冲液。
  21. 通过在65℃下加热使反面甲醛交联
  22. 向每个样品中加入18μl1M Tris-HCl pH6.5 0.5M EDTA和5μg蛋白酶K,并在48℃加热90分钟。
  23. 使用Qiagen PCR纯化试剂盒分离DNA
  24. 分离DNA的另一种方法是1×苯酚/氯仿提取,1×氯仿提取,O.N。使用2.5体积的无水乙醇,用30μl乙酸钾3M,pH 5.7和5μg糖原进行沉淀。用500μl70%乙醇洗涤沉淀两次并风干
  25. 重悬颗粒在35微升TE与0.1毫克/毫升核糖核酸酶。
  26. 使用每个反应2μlDNA进行常规PCR。例如,对于CCDN1基因座(-1039至-770bp)或CCDN1启动子(-235至-53bp)PCR,使用20μl含有1.5mM MgCl 2的H 2 O溶液0.2mM dNTP,0.25μMPRIMER F,0.25μMPRIMER R,0.5U /μlTAQ Pol,2μlDNA和2μlPCR缓冲液。使用33-35个循环的扩增(变性96℃20秒,退火50℃90秒,延伸69℃60秒)。在2%琼脂糖凝胶中在TBE 0.5%中运行

食谱

  1. 木炭葡聚糖剥离血清
    葡聚糖包被的木炭用于从血清中除去类固醇激素。 木炭/葡聚糖剥离的血清是可商购的,但是我们如Migliaccio等人所述制备血清。 (2011)。
  2. 用于MCF-7细胞(具有PR的DMEM)的无酚红的Dulbecco's改良培养基
    5%FBS
    2mM L-谷氨酰胺 100U/ml青霉素 - 链霉素 3.75 ng/ml氢化可的松
    6 ng/ml胰岛素
  3. 添加MCF-7细胞用炭的无酚红Dulbecco's改良培养基 (DMEM w/o PR)
    5%CSS
    2mM L-谷氨酰胺 100U/ml青霉素 - 链霉素 3.75 ng/ml氢化可的松
    6 ng/ml胰岛素
  4. 50x LAP(蛋白酶抑制剂混合物)
    将片剂溶解在1ml蒸馏的H 2 O中,并使用50×
  5. DTT解决方案
    10 mM DTT
    100mM Tris-HCl
    pH 9.5
  6. 缓冲区I
    0.25%Triton X-100 10 mM EDTA
    0.5mM EGTA 10 mM Hepes
    pH 6.5
  7. 缓冲区II
    200 mM NaCl
    1mM EDTA
    0.5mM EGTA 10 mM Hepes
    pH 6.5
  8. 缓冲液III或细胞裂解缓冲液
    1%SDS
    10 mM EDTA
    50mM Tris-HCl(pH8.1) 50x蛋白酶抑制剂混合物
  9. 缓冲区IV
    1%Triton X-100 2mM EDTA 150mM NaCl 20mM Tris-HCl(pH8.1)
  10. TSE I缓冲区
    0.1%SDS
    1%Triton X-100 2mM EDTA 20mM Tris-HCl(pH8.1) 150mM NaCl
  11. TSE II缓冲区
    0.1%SDS
    1%Triton X-100 2mM EDTA 20mM Tris-HCl(pH8.1) 500 mM NaCl
  12. TSE III缓冲区
    0.25 M LiCl
    1%NP40
    1%脱氧胆酸钠
    1mM EDTA
    10mM Tris-HCl(pH8.1)
  13. TE缓冲区
    20mM Tris-HCl(pH8.1) 1mM EDTA(pH8.0)
  14. 洗脱缓冲液
    1%SDS
    0.1 M NaHCO 3 3/v/v
  15. PCR缓冲区
    300 mM Tris-Base
    100 mM Hepes
    250 mM氯化钾
    200 mM谷氨酸钾
    20 mM DTT
    50%甘油
    过滤灭菌

致谢

这项工作由意大利癌症研究协会资助(A.I.R.C;批准号IG 5389)。 Pia Giovannelli获得了A.I.R.C.的奖学金支持。该协议改编自Castoria等人(2012)。

参考文献

  1. Castoria,G.,Giovannelli,P.,Lombardi,M.,De Rosa,C.,Giraldi,T.,de Falco,A.,Barone,MV,Abbondanza,C.,Migliaccio,A.and Auricchio, (2012)。 Src对雌二醇受体的酪氨酸磷酸化调节乳腺癌中其激素依赖性核输出和细胞周期进展细胞。 癌基因 31(46):4868-4877
  2. Lombardi,M.,Castoria,G.,Migliaccio,A.,Barone,MV,Di Stasio,R.,Ciociola,A.,Bottero,D.,Yamaguchi,H.,Appella,E。和Auricchio, 2008)。 乳腺癌细胞中雌激素受体和DNA合成的激素依赖性核输出。 em> J Cell Biol 182(2):327-340。
  3. Migliaccio,A.,Castoria,G.,Auricchio,F。(2011)。 分析细胞信号传导途径中的雄激素受体快速作用:受体/Src结合。 em> Methods Mol Biol  776:361-370。
  4. Yahata,T.,Shao,W.,Endoh,H.,Hur,J.,Coser,KR,Sun,H.,Ueda,Y.,Kato,S.,Isselbacher,KJ,Brown,M。和Shioda, T.(2001)。 CITED1 CBP/p300结合蛋白的雌激素依赖性转录的选择性共激活 。 Dev 15(19):2598-2612。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Giovannelli, P., Castoria, G. and Migliaccio, A. (2013). Estradiol Receptor (ER) Chromatin Immunoprecipitation in MCF-7 Cells. Bio-protocol 3(14): e831. DOI: 10.21769/BioProtoc.831.
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