Mettl14 hpa038002

Manufactured by Merck Group

METTL14 (HPA038002) is a lab equipment product manufactured by Merck Group. It is a component of the methyltransferase complex that plays a role in the regulation of gene expression. The core function of this product is to facilitate the methylation of specific RNA molecules, which is an important process in cellular biology. This description is based solely on the factual information available and does not make any interpretations or extrapolations about the intended use of the product.

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3 protocols using mettl14 hpa038002

Immunoblot Analysis of Protein Expression

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The immunoblot analysis was performed as described previously.^{15 (link)} Briefly, cells were lysed with RIPA buffer (20–188, Millipore) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermo Fisher Scientific). For the separation of nuclear and cytoplasmic proteins, cells were firstly lysed with cytoplasmic lysis buffer (Tris 10 mM, NaCl 10 mM, MgCl2 3 mM, Nonidet P-40 0.1%) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermo Fisher Scientific) for 3 min, and the supernatant was collected for the detection of cytoplasmic proteins. After three washes with cytoplasmic lysis buffer, the nuclei were lysed with RIPA buffer. Protein concentrations were measured with BCA protein assay kit (Thermo Fisher Scientific). ALKBH5 (HPA007196) and METTL14 (HPA038002) antibodies were from Sigma. YTHDF1 (17479-1-AP) and PTGER4 (66921-1-Ig) antibodies were from Proteintech. METTL3 (ab195352), FTO (ab92821), and Tenascin C (ab108930) antibodies were from Abcam. WNK1 (MA5-35466) and NLRP12 (PA5-89879) antibodies were from Invitrogen. GAPDH (M171-3) and YTHDF2 (RN123PW) antibodies were from MBL. Lamin A/C (4777 S) antibody was from CST. Goat anti-rabbit IgG-HRP (ZB-2301) and goat anti-mouse IgG-HRP (ZB-2305) antibodies were from ZSGB-BIO.

Liu Y., Song R., Zhao L., Lu Z., Li Y., Zhan X., Lu F., Yang J., Niu Y, & Cao X. (2022). m6A demethylase ALKBH5 is required for antibacterial innate defense by intrinsic motivation of neutrophil migration. Signal Transduction and Targeted Therapy, 7, 194.

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RNA Immunoprecipitation Analysis of METTL14

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RNA immunoprecipitation was performed as previously described (Rinn et al., 2007 (link)) with some modifications. Briefly, following ultraviolet crosslinking, MM6 cells were harvested and nuclear extracts were isolated and sonicated. One microgram of METTL14 (HPA038002, Sigma-Aldrich) antibody, eIF3A (#3411, CST) antibody, or rabbit control IgG (Thermo Fisher Scientific) was conjugated to Protein A/G Magnetic Beads (Thermo Fisher Scientific) by incubation at 4 °C for 4 hours, followed by 3× wash and incubation with pre-cleared nuclear extraction in RIP buffer (150 mM KCl, 25 mM Tris (pH 7.4), 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 1× protease inhibitor) at 4 °C overnight. After washing with RIP buffer for three times, beads were resuspended in 80 µL PBS, followed by DNA digestion at 37 °C for 15 min and incubation with 50 µg of Proteinase K (Thermo Fisher) at 37 °C for 15 min. Input and co-immunoprecipitated RNAs were recovered by TRIzol (Invitrogen) extraction and used for qPCR analyses using primers listed in Table S1.

Weng H., Huang H., Wu H., Qin X., Zhao B.S., Dong L., Shi H., Skibbe J., Shen C., Hu C., Sheng Y., Wang Y., Wunderlich M., Zhang B., Dore L.C., Su R., Deng X., Ferchen K., Li C., Sun M., Lu Z., Jiang X., Marcucci G., Mulloy J., Yang J., Qian Z., Wei M., He C, & Chen J. (2017). METTL14 Inhibits Hematopoietic Stem/Progenitor Differentiation and Promotes Leukemogenesis via mRNA m6A Modification. Cell stem cell, 22(2), 191-205.e9.

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Western Blot Analysis of RNA Methylation Proteins

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Western blots were performed using standard procedures. Briefly, 10–30 μg protein samples were separated on 4–12% polyacrylamide Bis-Tris gels (NP0336BOX, Invitrogen) and transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore). The blots were probed with METTL3- (15073-1-AP, Proteintech), METTL14- (HPA038002, Sigma), HNRNPG- (sc-14581 and sc-48796, Santa Cruz Biotechnology) or GAPDH- (A00192-40, Genscript) specific primary antibody, followed by rabbit anti-goat IgG-HRP (sc-2768, Santa Cruz Biotechnology) or goat anti-rabbit IgG-HRP (ab97051, Abcam) secondary antibody, and then visualized by enhanced chemoluminescence (RPN2109, GE Healthcare).

Liu N., Zhou K.I., Parisien M., Dai Q., Diatchenko L, & Pan T. (2017). N 6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein. Nucleic Acids Research, 45(10), 6051-6063.

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