A RIP assay detection kit (Merck Millipore) was used according to the manufacturer's instructions. RIP lysis buffer was prepared for cell lysis with 100 μl of RIP lysis buffer, 0.5 μl of protease inhibitor cocktail, and 0.25 μl of RNase inhibitor. Anti-METTL3 (1 : 50, E3F2A, Cell Signaling) was added to the resuspended beads, and the mixture was incubated for 30 min at room temperature. RIP immunoprecipitation buffer was prepared for RNA-binding protein immunoprecipitation with 860 μl of RIP wash buffer, 35 μl of 0.5 M EDTA, and 5 μl of RNase inhibitor. The RNA bound to the beads was purified and then reverse-transcribed into cDNA for qRT-PCR.
Anti mettl3
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-METTL3 is a primary antibody that recognizes the METTL3 protein. METTL3 is an enzyme that catalyzes the addition of methyl groups to RNA, a process known as N6-methyladenosine (m6A) methylation. This antibody can be used to detect and study the METTL3 protein in various biological samples.
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Lab products found in correlation
Anti mettl14, by Cell Signaling Technology (3 mentions)
Anti fto, by Cell Signaling Technology (3 mentions)
Anti alkbh5, by Cell Signaling Technology (2 mentions)
Anti wtap, by Cell Signaling Technology (2 mentions)
Click it nascent rna capture kit, by Thermo Fisher Scientific (1 mentions)
Anti mda5, by Cell Signaling Technology (1 mentions)
Anti mavs, by Santa Cruz Biotechnology (1 mentions)
Anti tbk1, by Cell Signaling Technology (1 mentions)
Anti p tbk1, by Cell Signaling Technology (1 mentions)
Anti alkbh5, by Abcam (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti rig 1, by Cell Signaling Technology (1 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Anti p irf3, by Cell Signaling Technology (1 mentions)
Anti irf3, by Cell Signaling Technology (1 mentions)
Epimark n6 methyladenosine enrichment kit, by New England Biolabs (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Anti eif4g1, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
8 protocols using anti mettl3
1
RNA-Binding Protein Immunoprecipitation Assay
2
RNA N6-methyladenosine Regulation Analysis
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Antibodies used in this study were summarized at the dilutions listed: anti‐METTL14, 1:1000, (IB; Cell Signaling Technology, #: 51104S), anti‐N6‐methyladenosine modifications of RNA and DNA (m6A), 1:2000, (dot blot; Synaptic Systems, #: 202 003); anti‐IRF3, 1:1000, (IB; Cell Signaling Technology, #: 4302S); anti‐pIRF3, 1:1000, (IB; Cell Signaling Technology, #: 4947S) anti‐TBK1, 1:1000, (IB; Cell Signaling Technology, #: 3504S); anti‐pTBK1, 1:1000, (IB; Cell Signaling Technology, #: 5483S); anti‐RIG‐I, 1:1000, (IB; Cell Signaling Technology, #: D14G6); anti‐MDA5, 1:1000, (IB; Cell Signaling Technology, #: D74E4); anti‐FTO, 1:1000 (IB; Cell Signaling Technology, #: D6Z8W); anti‐METTL3, 1:1000 (IB; Cell Signaling Technology, #: E3F2A); anti‐ALKBH5, 1:1000 (IB; Abcam, #: ab195377); anti‐MAVS, 1:500, (IB; Santa Cruz Biotechnology, #: sc‐365333); anti‐FLAG (M2), 1:1000, (IB; Sigma‐Aldrich, #: F1804); anti‐β‐actin, 1:2000, (IB; ZSGB‐BIO, #:TA‐09). CHX (HY‐12320), actinomycin‐D, (HY‐17559), were obtained from MedChemExpress (MCE, NJ, USA); PMA (P1585) and Dynabeads mRNA Purification Kit (#: 61006) were purchased from Invitrogen; Click‐iT nascent RNA capture kit (#: C10365) was purchased from Life Technologies; EpiMark N6‐Methyladenosine Enrichment Kit (E1610S) was purchased from New England Biolabs.
3
Protein Expression Analysis Using Western Blot
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Cells were lysed using RIPA buffer (CW2333S; CWbio, Beijing, China) supplemented with a protease inhibitor cocktail set I (539131; Millipore, Burlington, MA, USA). Western blot was performed according to a standard protocol as previously described (15 (link)). Primary antibodies included anti-mTOR (#2983), anti-METTL3 (#86132), anti-WTAP (#41934), anti-FTO (#31687), anti-ALKBH5 (#80283), anti-LC3B (#4599), anti-eIF4G1 (#2858) (1:1 000, Cell Signaling Technology, Danvers, MA, USA), anti-METTL14 (#ab220030, 1:1 000, Abcam, Cambridge, UK), and anti-GAPDH (#G5262, 1:1 000, Sigma-Aldrich, St. Louis, MO, USA). Western blot band intensity was quantified using ImageJ 1.8.0 (https://imagej.nih.gov/ij/download.html ).
4
Myocardial Tissue Histochemical Analysis
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Myocardial samples were incubated in paraformaldehyde (4%) prior to embedment in paraffin. Samples were sliced into sequential pieces (5-μm in thickness) and were stained with Hematoxylin and eosin (HE, Cat No. C0105 M, Beyotime Biotechnology) or Masson's Trichrome (Cat No. ab150686, Abcam). For immunohistochemical staining, sample slices were exposed overnight to anti-METTL3 (1:200, Cat No. 86132, Cell Signaling Technology) or anti- 4-hydroxynonenal (4-HNE, 1:100, Cat No. ab46545, Abcam) antibody, prior to nurturing with a secondary antibody. Images were captured through a digital microscope and were processed using Image J (v1.34S).
5
Western Blot Analysis of Epigenetic Regulators
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Samples (tissues and cells) were collected and lysed in a RIPA lysis buffer (Cat No. P0013B, Beyotime Biotechnology). Protein samples were isolated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) prior to transfer onto 0.22-μm PVDF membranes. Following 5% bovine serum albumin (BSA) blocking, PVDF membranes were cultivated with primary antibodies, including anti-METTL3 (1:1000, Cat No. 96391, Cell Signaling Technology), anti-METTL14 (1:1000, Cat No. 51104, Cell Signaling Technology), anti-WTAP (1:1000, Cat No. 56501, Cell Signaling Technology), anti-FTO (1:1000, Cat No. 51104, Cell Signaling Technology), anti-ALKBH5 (1:1000, Cat No. 80283, Cell Signaling Technology), anti-c-Jun (1:1000, Cat No. 9165, Cell Signaling Technology), anti-GPX4 (1:1000, ab125066, Abcam); anti-SLC7A11 (1:1000, ab307601, Abcam), anti-TFRC (1:1000, Cat No. ab109259, Abcam), anti-IGF2BP2 (1:1000, Cat No. 14672, Cell Signaling Technology), anti-GAPDH (1:2000, ab9485, Abcam), anti-Tubulin (1:1000, Cat No. 2144, Cell Signaling Technology).
6
Western Blot Analysis of Protein Expression
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Cells were lysed in RIPA buffer supplemented with protease inhibitors cocktails (Roche) and 20 μg of total protein were subjected to 12% SDS polyacrylamide gel electrophoresis. The samples were transferred to Amersham HybondTM -P membrane (GE Healthcare) and blotted using anti-F (SantaCruz, 1:500); anti-Flag (BioLegend, 1:5,000); anti-GFP (SantaCruz, 1:5,000), anti-METTL3 (Cell Signaling Technology, 1:1,000) and anti-GAPDH (SantaCruz, 1:5,000). A corresponding horseradish peroxidase (HRP)-conjugated antibody (Jackson ImmunoResearch, 1:10,000) was used as a secondary antibody. The membranes were analyzed with the ECL substrate (Cyanagen) using a MiniHD9 scanner (Uvitec).
7
Western Blot Analysis of m6A Regulators
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Briefly, the extracted proteins were separated by 10% SDS-PAGE and shifted to a PVDF membrane (Sigma Aldrich, St. Louis, MO, USA), followed by incubation with primary antibodies against METTL3, METTL14, ALKBH5, FTO, YTHDF1, YTHDF2, and YTHDF3. The probed membranes were then washed with TBST and incubated with corresponding secondary antibodies. After TBST washing, bands were visualized with ECL kit (Beyotime, Shanghai, China) using chemiluminescence image analysis system. The primary antibodies anti-METTL3 and anti-METTL14 were obtained from Cell Signaling Technology, Danvers, MA, anti-YTHDF1 and anti-YTHDF2 were obtained from Abcam, Cambridge, UK, and anti-YTHDF3 and anti-GAPDH were obtained from Absin, Shanghai, China while anti-ALKBH5 and anti-FTO (Rabbit polyclonal antibodies) were self-prepared and stored in the lab.
8
Antibodies for METTL3 and COL3A1 Analysis
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The primary antibodies for western blot, anti-METTL3 (#96391) were sourced from Cell Signaling Technology (MA, USA), anti-COL3A1 (sc-514601) was sourced from Santa Cruz (CA, USA), anti-α-tubulin (ab7291) was sourced from Abcam (CA, USA). HRP-conjugated goat anti-mouse/rabbit secondary antibodies (ZDR-5306/5307) were sourced from ZSBIO (Beijing, China). The antibodies for immunohistochemistry, anti-METTL3 (ab195352) were sourced from Abcam (CA, USA), and anti-COL3A1 (sc-166316) was sourced from Santa Cruz (CA, USA).
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