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Alkbh5

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ALKBH5 is an enzyme that catalyzes the demethylation of N6-methyladenosine (m6A) in RNA. It is involved in the regulation of RNA metabolism and plays a role in various biological processes.

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Lab products found in correlation

Znf333, by Thermo Fisher Scientific (3 mentions) Gapdh, by Abcam (3 mentions) β actin, by Cell Signaling Technology (3 mentions) Ythdf2, by Proteintech (2 mentions) C myc clone 9e10, by Santa Cruz Biotechnology (2 mentions) Mettl3, by Cell Signaling Technology (2 mentions) O glcnac clone rl2, by Thermo Fisher Scientific (2 mentions) D2hgdh, by Proteintech (2 mentions) L2hgdh, by Proteintech (2 mentions) Fto c 3, by Santa Cruz Biotechnology (2 mentions) Mettl14, by Cell Signaling Technology (2 mentions) Lamin a, by Fortis Life Sciences (2 mentions) β actin, by Merck Group (2 mentions) Gapdh, by ABclonal (2 mentions) α sma, by Abcam (2 mentions) Cd133, by Abcam (1 mentions) Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Caspase 3, by Abcam (1 mentions) P mtor, by Abcam (1 mentions)

Spelling variants of this product

Anti-ALKBH5 (11 citations) Anti-ALKBH5 antibody (3 citations)

20 protocols using alkbh5

1

Comprehensive Molecular Profiling Protocol

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Lipofectamine 2000 transfection reagents and total RNA extraction agent (TRIzol) were purchased from Invitrogen (Grand Island, NY, USA). Antibodies against Ki67, DDIT4, ALKBH5, Mettle3, HuR, Caspase-3, mTOR, p-mTOR, p-ULK, p-Ps70S6K, CD133, and CD44 were obtained from Abcam (Cambridge, MA, USA), whereas those against OCT4, SOX2, and EpCAM were obtained from Cell Signaling Technology (Danvers, MA, USA). UPF1 antibodies were purchased from Becton Dickinson. Unless indicated otherwise, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Details of antibody information are provided in Supplementary materials.
Zhang Y., Liu X., Wang Y., Lai S., Wang Z., Yang Y., Liu W., Wang H, & Tang B. (2022). The m6A demethylase ALKBH5-mediated upregulation of DDIT4-AS1 maintains pancreatic cancer stemness and suppresses chemosensitivity by activating the mTOR pathway. Molecular Cancer, 21, 174.
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2

Protein Expression Analysis of m6A Regulators

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The cells were extracted with protein lysis buffer (Beyotime, China) supplemented with protease inhibitor cocktail. Protein concentration was determined using the BCA Kit (Beyotime, China). Proteins (25–35 μg) were separated on a 10% polyacrylamide precast SDS gel (Bio-Rad) followed by blotting on PVDF membranes (Millipore Billerica, MA, USA). The membranes were probed with the following antibodies against: Zfp217 (Abcam, USA, #48133), METTL3 (Abcam, #195352), ALKBH5 (Abcam, #69325), PPARγ (Cell Signaling Technology, USA, #C26H12), aP2 (Cell Signaling Technology, #D25B3), FTO (Santa cruz, USA, #sc-271713), YTHDF2 (Proteintech, China, #24744-1-AP), β-actin (Abclonal, China, #AC026), LMNB1 (Abclonal, #A1910) and Tublin (Abclonal, #AC021). Secondary antibodies and detection were according to description previously. For IP experiments, cells were lysed in IP buffer (Beyotime, China) and incubated with antibodies followed by the pull-down with protein A/G beads for subsequent western blot analysis.
Song T., Yang Y., Wei H., Xie X., Lu J., Zeng Q., Peng J., Zhou Y., Jiang S, & Peng J. (2019). Zfp217 mediates m6A mRNA methylation to orchestrate transcriptional and post-transcriptional regulation to promote adipogenic differentiation. Nucleic Acids Research, 47(12), 6130-6144.
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3

Immunoblotting Analysis of Cell Signaling Proteins

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Cell lysates were immunoblotted with primary antibodies targeting ALKBH5 (Abcam), ZNF333 (Invitrogen), CDX2 (Abcam), VIL1 (Abcam), KLF4 (Abcam), YTHDF2 (Abcam), p65 (CST), p-p65 (CST), FLAG (CST), and GAPDH (Abcam). The intensity of the fluorescence was detected using a chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA), and quantified using Quantity One software (Bio-Rad, USA).
Yue B., Cui R., Zheng R., Jin W., Song C., Bao T., Wang M., Yu F, & Zhao E. (2021). Essential role of ALKBH5-mediated RNA demethylation modification in bile acid-induced gastric intestinal metaplasia. Molecular Therapy. Nucleic Acids, 26, 458-472.
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4

Western Blot Analysis of Protein Targets

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Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and ruptured with RIPA buffer (Pierce, Rockford, IL) containing 5 mM EDTA, PMSF, cocktail proteinase inhibitors, and phosphatase inhibitor cocktail. Cell extracts were centrifuged at 12000 × g for 15 min and the supernatants were then collected. Cell lysates were resolved by SDS-PAGE and transferred onto PVDF membranes which were blocked with 5% non-fat milk (Bio-Rad) in Tris-buffered saline containing 0.1% Tween 20 for 1 hour and incubated sequentially with primary and secondary antibodies and detected by immunoblotting with the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). Antibodies used for Western blotting were as follows: ALKBH5 (ab195377, abcam), ALKBH5 (HPA007196, Sigma-Aldrich), TACC3 (sc-376883, Santa Cruz Biotechnology), Flag (F3165, Sigma-Aldrich), c-Myc (9402S, Cell Signaling Technology), P21 (2947S, Cell Signaling Technology) GAPDH (sc-47724, Santa Cruz Biotechnology), β-Actin (3700, Cell Signaling Technology), Vinculin (sc-25336, Santa Cruz Biotechnology). GAPDH or β-Actin or Vinculin was used as a loading control.
Shen C., Sheng Y., Zhu A., Robinson S., Jiang X., Dong L., Chen H., Su R., Yin Z., Li W., Deng X., Chen Y., Hu Y.C., Weng H., Huang H., Prince E., Cogle C.R., Sun M., Zhang B., Chen C.W., Marcucci G., He C., Qian Z, & Chen J. (2020). M6A demethylase ALKBH5 selectively promotes tumorigenesis and cancer stem cell self-renewal in acute myeloid leukemia. Cell stem cell, 27(1), 64-80.e9.
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5

Comprehensive Protein Analysis in Metabolic Disorders

Cited 2 times
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Whole-cell lysates (WCL) were examined as we described36 (link). Nuclear and cytoplasmic proteins were extracted and analyzed as we recently reported5 (link). The following antibodies were used: c-MYC (clone 9E10; #sc-40, Santa Cruz Biotechnology), IDH2 (#ab55271, Abcam), OGT (#24083, Cell Signaling Technology), OGA (Cat #14711–1-AP, Proteintech), O-GlcNAc (clone RL2, #MA1–072, Thermo-Fisher Scientific), D2HGDH (#13895–1-AP, Proteintech), L2HGDH (#15707–1-AP, Proteintech), ALKBH5 (# ab69325, Abcam), FTO (c-3, #sc-271713, Santa Cruz Biotechnology), TET1 (#SAB2700730, Sigma-Aldrich), TET2 (#18950, Cell Signaling Technology), TET3 (#: ABS463, EMD Millipore Corporation), METTL3, METTL14, WTAP (# 69391, # 51104 and #56501, all from Cell Signaling Technology), β-actin (# A2228, Sigma-Aldrich), Lamin A (#A303–433A-M, Bethyl Laboratories), Tubulin (#62204 Invitrogen). PVDF membranes were stripped and re-probed with relevant antibodies for loading control, as reported37 (link).
Lin A.P., Qiu Z., Ethiraj P., Sasi B., Jaafar C., Rakheja D, & Aguiar R.C. (2022). MYC, mitochondrial metabolism and O-GlcNAcylation converge to modulate the activity and subcellular localization of DNA and RNA demethylases. Leukemia, 36(4), 1150-1159.
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6

Comprehensive Protein Analysis in Metabolic Disorders

Cited 2 times
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Whole-cell lysates (WCL) were examined as we described36 (link). Nuclear and cytoplasmic proteins were extracted and analyzed as we recently reported5 (link). The following antibodies were used: c-MYC (clone 9E10; #sc-40, Santa Cruz Biotechnology), IDH2 (#ab55271, Abcam), OGT (#24083, Cell Signaling Technology), OGA (Cat #14711–1-AP, Proteintech), O-GlcNAc (clone RL2, #MA1–072, Thermo-Fisher Scientific), D2HGDH (#13895–1-AP, Proteintech), L2HGDH (#15707–1-AP, Proteintech), ALKBH5 (# ab69325, Abcam), FTO (c-3, #sc-271713, Santa Cruz Biotechnology), TET1 (#SAB2700730, Sigma-Aldrich), TET2 (#18950, Cell Signaling Technology), TET3 (#: ABS463, EMD Millipore Corporation), METTL3, METTL14, WTAP (# 69391, # 51104 and #56501, all from Cell Signaling Technology), β-actin (# A2228, Sigma-Aldrich), Lamin A (#A303–433A-M, Bethyl Laboratories), Tubulin (#62204 Invitrogen). PVDF membranes were stripped and re-probed with relevant antibodies for loading control, as reported37 (link).
Lin A.P., Qiu Z., Ethiraj P., Sasi B., Jaafar C., Rakheja D, & Aguiar R.C. (2022). MYC, mitochondrial metabolism and O-GlcNAcylation converge to modulate the activity and subcellular localization of DNA and RNA demethylases. Leukemia, 36(4), 1150-1159.
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7

Quantifying Protein Expression via IHC

Cited 1 time
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The IHC assay was conducted with antibodies against human ALKBH5 (Abcam), ZNF333 (Invitrogen), CYLD (Abcam), CDX2 (Abcam), or p-p65 (CST). The final staining was divided into three grades (negative, weak, and strong) based on the staining intensity and area.27 (link) The staining intensity was scored as 0 (no staining), 1 (weak), or 2 (strong). The staining area was scored as follows: 0 (≤10% positive staining), 1 (11%–25% positive staining), 2 (26%–50% positive staining), 3 (51%–75% positive staining), and 4 (≥75% positive staining). Staining intensity and staining area were summed up to give a final total score index: an overall score of ≤3 was defined as negative expression, of >3–≤6 as weak expression, and of >6 as strong expression. It was also divided into high-expression (strong staining) and low-expression (negative and weak staining) groups.
Yue B., Cui R., Zheng R., Jin W., Song C., Bao T., Wang M., Yu F, & Zhao E. (2021). Essential role of ALKBH5-mediated RNA demethylation modification in bile acid-induced gastric intestinal metaplasia. Molecular Therapy. Nucleic Acids, 26, 458-472.
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8

Protein Extraction and Western Blot

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We used the Total Protein Extraction Kit (Keygentec, China) to extract cells or frozen tissues. BCA Protein Assay kit (Pierce, USA) was used to calculate the protein concentration. Western blot analysis followed a standard procedure. The primary antibodies (ALKBH5, ITPA, GAPDH, and β-actin) were obtained from Abcam, USA. The anti-mouse and anti-rabbit secondary antibodies were obtained from Cell Signaling Technology, USA.
Li R., Wu X., Xue K., Feng D., Li J, & Li J. (2023). RNA demethylase ALKBH5 promotes tumorigenesis of t (8;21) acute myeloid leukemia via ITPA m6A modification. Biomarker Research, 11, 30.
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9

Protein Lysis, Quantification, and Western Blotting

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Cells were lysed in RIPA buffer (P0013B, Beyotime, China) containing the complete cocktail of protease inhibitors (#11836153001, Roche, Switzerland). Protein concentrations were determined with the BCA protein assay kit (P0011, Beyotime, China). Proteins were separated by 4-20% SDS-PAGE, transferred to PVDF film and blotted with antibodies at 4 °C overnight. Secondary antibodies were pre-labelled at room temperature for 1 h. The PVDF film with the target protein was exposed in the visualizer (AI800, GE, USA). Antibodies were purchased from the following: ALKBH5 (ab195377, 1:1000, Abcam), Caspase-3 (A19654, 1:1000, Abclonal), SQSTM1/p62 (A19700, 1:1000, Abclonal), GAPDH (A19056, 1:10000, Abclonal).
Wang H., Zhao S., Liu H., Liu Y., Zhang Z., Zhou Z., Wang P., Qi S, & Xie J. (2024). ALKBH5 facilitates the progression of skin cutaneous melanoma via mediating ABCA1 demethylation and modulating autophagy in an m6A-dependent manner. International Journal of Biological Sciences, 20(5), 1729-1743.
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10

Probing IGF1 Signaling Pathways

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IGF1 (AFL291) was obtained from R&D System Company (Minneapolis, USA). Antibodies used for IP, immunoblot (IB) analysis and immunofluorescence (IF) were as follows: m1A antibody (D345-3, 1:100 for IP) was from MBL Life Science (Japan). Horse radish peroxidase-coupled secondary antibodies (ab6721, 1:1000 for IB), IGF1R (ab182048, 1:1000 for IB), ALKBH5 (ab69325, 1:1000 for IB), and YTHDF3 (ab83716, 1:1000 for IB) were from Abcam (Cambridge, UK). β-Actin (4970, 1:1000 for IB), p-ERK (4370, 1:1000), and p-p38 (4511, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA). The Flag tags (F7425, 1:1000 for IB) as well as the agarose were used for IP; LPS (L2630) was from Sigma-Aldrich (St. Louis, MO, USA). DAPI (4′,6-diamidino-2-phenylindole) (62247, 1:1000 for IF) Alexa Fluor® 546 (A-11071, 1:500 for IF) and Protein A/G Magnetic Beads (88802) were from Thermo Fisher Scientific.
Zheng Q., Gan H., Yang F., Yao Y., Hao F., Hong L, & Jin L. (2020). Cytoplasmic m1A reader YTHDF3 inhibits trophoblast invasion by downregulation of m1A-methylated IGF1R. Cell Discovery, 6, 12.
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