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Immunoprecipitation of Acetyl-lysine and Western Blotting of Long-chain acyl-CoA Dehydrogenases and Beta-hydroxyacyl-CoA Dehydrogenase in Palmitic Acid Treated Human Renal Tubular Epithelial Cells

棕榈酸治疗的人肾小管上皮细胞中乙酰赖氨酸的免疫沉淀和长链酰基辅酶A脱氢酶和β-羟酰基辅酶A脱氢酶的蛋白质印迹

TL Tingting Lv
SZ Suwei Zhu
YM Yuan Ma
HF Hong Feng
QW Qiang Wan

发布: 2020年09月20日第10卷第18期 DOI: 10.21769/BioProtoc.3765 浏览次数: 5009

评审: Xiaoyi ZhengYing ShiJake J. Wen

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Abstract

As one of the main energy metabolism organs, kidney has been proved to have high energy requirements and are more inclined to fatty acid metabolism as the main energy source. Long-chain acyl-CoA dehydrogenases (LCAD) and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD), key enzymes involved in fatty acid oxidation, has been identified as the substrate of acetyltransferase GCN5L1 and deacetylase Sirt3. Acetylation levels of LCAD and beta-HAD regulate its enzymes activity and thus affect fatty acid oxidation rate. Moreover, immunoprecipitation is a key assay for the detection of LCAD and beta-HAD acetylation levels. Here we describe a protocol of immunoprecipitation of acetyl-lysine and western blotting of LCAD and beta-HAD in palmitic acid treated HK-2 cells (human renal tubular epithelial cells). The scheme provides the readers with clear steps so that this method could be applied to detect the acetylation level of various proteins.

Keywords: Immunoprecipitation (免疫沉淀反应) search Acetylation (乙酰化作用) search LCAD (长链酰基辅酶A脱氢酶) search Beta-HAD (β-羟酰基辅酶A脱氢酶) search Palmitic acid (PA) (棕榈酸) search HK-2 cells (HK-2 细胞) search

Background

Post-translational modifications (PTMs) enable the cell with a highly dynamic mechanism for regulation of cellular pathways (Zhao et al., 2010). Acetylation has emerged as one of the major post-translational protein modifications. Accumulating evidences indicate that acetylation rivals phosphorylation as a regulatory modification in mitochondria (Henriksen et al., 2012). Over 60% of mitochondrial proteins are acetylated, being of notice, which were involved in energy metabolism such as tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), fatty acid oxidation and amino acid metabolism (Hirschey et al., 2010; Hebert et al., 2013). Acetylation is also considered to be one of the main adaptive mechanisms for mitochondria in sensing nutritional challenges. Under the condition of excess energy supplement, mitochondrial proteins are hyperacetylated. Insufficient energy availability leads to a decreased acetylation level of mitochondrial proteins (Schwer et al., 2009). A large number of mitochondrial metabolic enzymes are acetylated in diabetes, including the FAO enzymes LCAD and beta-HAD (Kosanam, 2014). Dharendra et al. indicated that the upregulation of acetylation status of mitochondrial fatty acid oxidation enzymes promoted fatty acid oxidation in the heart of high fat diet mice (Thapa et al., 2018). Our previous study also found that the acetylation levels of FAO enzymes LCAD and β-HAD were enhanced in HK-2 cells treated with palmitic acid (PA) (Lv et al., 2019). Protein lysine acetylation refers to covalent addition of acetyl group to lysine residue (N-lysine acetylation). At present, since there are no acetylated antibodies of LCAD and HAD, the detection of LCAD and HAD acetylation levels mainly depends on immunoprecipitation analysis which mainly concentrates the acetylated proteins by using acetyl-lysine antibody and then detects the content of acetylated LCAD and acetylated HAD by using LCAD antibody and HAD antibody respectively (Figure 1).


Figure 1. Flowchart of IP

Materials and Reagents

  1. 100 mm x 20 mm cell culture dishes (Coring, catalog number: 430167 )
  2. 1.5 ml microcentrifuge tube
  3. Disposable syringe filters (Millipore, catalog number: SLGP033RB )
  4. Polyvinylidene fluoride (PVDF) Transfer Membrane (Thermo Scientific, catalog number: 88518 )
  5. HK-2 cells (ATCC, catalog number: CRL-2190 )
  6. Pierce Crosslink Immunoprecipitation Kit (Thermo Scientific, catalog number: 26147 )
  7. Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, catalog number: 11885084 )
  8. Fetal bovine serum (FBS) (Gibco, catalog number: 10099141C )
  9. Palmitic acid (PA) (Sigma, catalog number: P5585 )
  10. Albumin Bovine (BSA) (Solarbio, catalog number: A8850 )
  11. Halt Protease and Phosphatase inhibitor cocktail (Thermo Scientific, catalog number: 78442 )
  12. Phosphate buffered saline (PBS) (Solarbio, catalog number: P1020 )
  13. Mouse-Acetyl-lysine antibody (Abcam, catalog number: ab22550 )
  14. Rabbit-LCAD antibody (Proteintech, catalog number: 17526-1-AP )
  15. Rabbit-beta-HAD antibody (Sigma, catalog number: HPA037539 )
  16. Rabbit-COXIV antibody (Proteintech, catalog number: 11242-1-AP )
  17. Secondary anti-rabbit IgG HRP-conjugated antibody
  18. Glycine (Solarbio, catalog number: G8200 )
  19. Sodium dodecyl sulfate (SDS) (Solarbio, catalog number: S8010 )
  20. Ammonium persulfate (APS) (Solarbio, catalog number: A8090 )
  21. N,N,N',N'-Tetramethylethylenediamine (TEMED) (Invitrogen, catalog number: 15524010 )
  22. Tween-20 (Solarbio, catalog number: T8220 )
  23. Tris (Solarbio, catalog number: T8060 )
  24. 1.5 M Tris(hydroxymethyl)aminomethane (Tris-HCl) (pH 8.8) (Solarbio, catalog number: T1010 )
  25. 1.0 M Tris(hydroxymethyl)aminomethane (Tris-HCl ) (pH 6.8) (Solarbio, catalog number: T1020 )
  26. 30% Acrylamide (Solarbio, catalog number: A1010 )
  27. Bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, catalog number: P0012S )
  28. Immobilon Western Chemiluminescent HRP Substrate (Millipore, catalog number: WBKLS0050 )
  29. Methanol (Tianjin Kaitong, China)
  30. Sodium chloride (Tianjin Kaitong, China)
  31. 12% Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separating gels (10 ml) (see Recipes)
  32. SDS-PAGE stacking gels (4 ml) (see Recipes)
  33. 1x TBST (Tris-HCl, NaCl, Tween 20) (see Recipes)
  34. 2.5% BSA (see Recipes)
  35. 10 mM PA (PA stock solution) (see Recipes)
  36. 200 μM PA (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, catalog number: 5810R )
  2. P1000 pipette (Eppendorf, catalog number: 3111000858 )
  3. P200 pipette (Eppendorf, catalog number: 3111000840 )
  4. P20 pipette (Eppendorf, catalog number: 3111000823 )
  5. P10 pipette (Eppendorf, catalog number: 3111000815 )
  6. Rotator
  7. Benchtop rocker (HEIDOLPH, catalog number: Titramax 101 )
  8. Spectrophotometer
  9. Blot and gel imager (General Electric Company, catalog number: Amersham Imager 680 )

Procedure

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文章信息

版权信息

© 2020 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Lv, T., Zhu, S., Ma, Y., Feng, H. and Wan, Q. (2020). Immunoprecipitation of Acetyl-lysine and Western Blotting of Long-chain acyl-CoA Dehydrogenases and Beta-hydroxyacyl-CoA Dehydrogenase in Palmitic Acid Treated Human Renal Tubular Epithelial Cells. Bio-protocol 10(18): e3765. DOI: 10.21769/BioProtoc.3765.

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分类

细胞生物学 > 细胞新陈代谢 > 其它化合物

细胞生物学 > 基于细胞的分析方法 > 酶学测定

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