Nsun2

Manufactured by Proteintech

Sourced in United States

NSUN2 is a protein-coding gene that is responsible for the synthesis of the enzyme N6-methyltransferase. This enzyme is involved in the methylation of specific nucleotides within ribosomal RNA, which is a crucial step in the process of protein synthesis.

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Lab products found in correlation

Gapdh, by Proteintech (2 mentions) Gapdh, by Transgene (1 mentions) Bicinchoninic acid (bca), by Beyotime (1 mentions) Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions) Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions) Stat3 124h6, by Cell Signaling Technology (1 mentions) β actin, by Merck Group (1 mentions) Chemidoc touch imaging system, by Bio-Rad (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Gapdh, by Merck Group (1 mentions) P0013, by Beyotime (1 mentions)

5 protocols using nsun2

Protein Extraction and Western Blotting

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HAP1 cells were collected and lysed by RIPA buffer (150 mM NaCl, 50 mM Tris, 1% EDTA, 1% NP40, 0.1% SDS). Lysate was incubated at 4°C for 30 min, then sonicated with 10 cycles (30 s On /30 s Off), and then centrifuged at 15,000 g for 15 min at 4°C. The total protein concentration was measured by BCA (Beyotime). 60 µg total protein was loaded and separated on the 10% SDS–polyacrylamide gel. The protein on the gel was transfected to the polyvinylidene difluoride membranes (Immobilon‐P). The membrane was incubated with primary antibody and horseradish peroxidase–conjugated secondary antibody, and then proteins were detected using the Pierce™ ECL Western Blotting Substrate (Thermo) by BIO‐RAD ChemiDoc™ XRS+ system. The following antibodies were used for western blotting: NSUN2 (Proteintech), NSUN6 (Proteintech), GAPDH (TransGen Biotech), goat anti‐mouse IgG‐HRP (Santa cruz biotechnology), and goat anti‐rabbit IgG‐HRP (Santa cruz biotechnology).

Fang L., Wang W., Li G., Zhang L., Li J., Gan D., Yang J., Tang Y., Ding Z., Zhang M., Zhang W., Deng D., Song Z., Zhu Q., Cui H., Hu Y, & Chen W. (2020). CIGAR‐seq, a CRISPR/Cas‐based method for unbiased screening of novel mRNA modification regulators. Molecular Systems Biology, 16(11), e10025.

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Western Blot Analysis of Epigenetic Regulators

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Whole‐cell lysates were prepared using modified radioimmunoprecipitation assay (RIPA) buffer as described previously [28 (link)]. The following primary antibodies were used for Western blotting at 1 : 1000 dilution: NSUN5 (H‐10) (Santa Cruz Biotechnology, Dallas, TX, USA; Cat# sc‐376 147, RRID: AB_10989978), NSUN2 (Proteintech, Rosemont, IL, USA; Cat# 20854‐1‐AP, RRID: AB_10693629), STAT3 (124H6) (Cell Signaling Technology, Danverss, MA, USA; Cat#9139, RRID: AB_331757), β‐Actin (Sigma‐Aldrich, Oakville, ON, Canada; Cat# A5441, RRID: AB_476744), and Tubulin (Abcam, Toronto, ON, Canada, ab59680).

Zhou J., Kong Y.S., Vincent K.M., Dieters‐Castator D., Bukhari A.B., Glubrecht D., Liu R., Quilty D., Findlay S.D., Huang X., Xu Z., Yang R.Z., Zhang L., Tang E., Lajoie G., Eisenstat D.D., Gamper A.M., Fahlman R., Godbout R., Postovit L, & Fu Y. (2023). RNA cytosine methyltransferase NSUN5 promotes protein synthesis and tumorigenic phenotypes in glioblastoma. Molecular Oncology, 17(9), 1763-1783.

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Immunoblotting Analysis of Protein Modifications

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Cells were harvested and dissolved in RIPA lysis buffer and a protease-inhibitor cocktail. Whole-cell lysates were subjected to SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocking and incubating with specific primary and secondary antibodies, the proteins on the membranes were visualized using the Bio-Rad ChemiDoc^® Touch Imaging System (Bio-Rad).
The antibodies used included HA-Tag (1:1000, CST), NSUN2 (1:5000, Proteintech), SUMO-2/3 (1:1000, CST, #4971), GAPDH (1:1000, Proteintech; 60004-1-Ig), α-Tubulin (1:1000, CST; #3873), and Lamin A/C (1:1000, CST; #4777).

Hu Y., Chen C., Tong X., Chen S., Hu X., Pan B., Sun X., Chen Z., Shi X., Hu Y., Shen X., Xue X, & Lu M. (2021). NSUN2 modified by SUMO-2/3 promotes gastric cancer progression and regulates mRNA m5C methylation. Cell Death & Disease, 12(9), 842.

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Western Blot Analysis of NSUN2 Protein

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Cells were lysed with lysis buffer (NCM Biotech, Suzhou, China) supplemented with cocktail. A total of 30–40 μg of protein was separated by 10% SDS-polyacrylamide gel electrophoresis, and the PVDF membranes (Millipore, Billerica, USA) were blocked with 5% nonfat milk. Then, the membranes were incubated with the following primary antibodies overnight at 4°C: NSUN2 (1:1000) and GAPDH (1:10000) (Proteintech Group, Inc., Wuhan, China), and with secondary antibody for 1 h at 37°C. Finally, bands on immunoblots were detected with western blotting substrate (Share-Bio, Shanghai, China).

Zheng L., Li M., Wei J., Chen S., Xue C., Duan Y., Tang F., Li G., Xiong W., She K., Deng H, & Zhou M. (2023). NOP2/Sun RNA methyltransferase 2 is a potential pan-cancer prognostic biomarker and is related to immunity. PLOS ONE, 18(9), e0292212.

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Western Blot Analysis of Viral Proteins

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Cells were lysed on ice for 30 min with lysis buffer (P0013, Beyotime), centrifuged at 14,000 rpm for 10 min, and denatured at 100 °C. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The following steps were performed as described previously [51 (link)]. The primary antibodies included Flag (cat. no. F1804-1 MG; Sigma-Aldrich), GAPDH (cat. no. 60004-1-lg; Proteintech, Rosemont, IL, USA), NSUN2 (cat. no. 20854-1-AP; Proteintech, Rosemont, IL, USA), DNMT2 (cat. no. 19221-1-AP; Proteintech, Rosemont, IL, USA), ALYREF (cat. no. 16690-1-AP; Proteintech, Rosemont, IL, USA), HA (cat. no. 51064-2-AP; Proteintech, Rosemont, IL, USA), and PreS2 (cat. No. sc-23944; Santa Cruz Biotechnology, Dallas, TX, USA). The anti-HBsAg antibody was gifted by Dr. Bing Yan from the Wuhan Institute of Virology, CAS. At least three independent experiments for each western blot were performed. Original western blots are shown in Supplementary Material.

Ding S., Liu H., Liu L., Ma L., Chen Z., Zhu M., Liu L., Zhang X., Hao H., Zuo L., Yang J., Wu X., Zhou P., Huang F., Zhu F, & Guan W. (2024). Epigenetic addition of m5C to HBV transcripts promotes viral replication and evasion of innate antiviral responses. Cell Death & Disease, 15(1), 39.

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