Our western blotting assays were performed strictly following the standard protocols of our laboratory. Firstly, mouse heart tissues were ground using the tissue crusher with the tissue lysate, and lysates were prepared with RIPA lysis buffer from Beyotime Biotechnology, where protease inhibitor were added. Blots were screened using specific antibodies: GAPDH (5174, 1:10000; CST), Mettl3 (ab195352, 1:1000; Abcam), FTO (ab92821, 1:1000; Abcam), ALKBH5 (ab69325, 1:1000; Abcam), Mettl14 (HPA038002, 1:1000; Sigma), and m6A (ab208577, 1:500; Abcam).
Ab92821
Manufactured by Abcam
Sourced in United States
Ab92821 is an antibody product offered by Abcam. It is a primary antibody designed for use in various laboratory applications. The core function of this product is to bind to and detect the target antigen. Further details about the intended use or performance of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab195352, by Abcam (5 mentions)
Hpa038002, by Merck Group (5 mentions)
Hpa007196, by Merck Group (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Ab208577, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ab103328, by Abcam (3 mentions)
Ab99080, by Abcam (2 mentions)
Ab4103, by Abcam (2 mentions)
Ab133836, by Abcam (2 mentions)
Ab176846, by Abcam (2 mentions)
Nbp1 83040, by Novus Biologicals (2 mentions)
P0067, by Abcam (2 mentions)
Ab195377, by Abcam (2 mentions)
Ab69325, by Abcam (1 mentions)
Ab190886, by Abcam (1 mentions)
A2228, by Merck Group (1 mentions)
Ab220162, by Abcam (1 mentions)
Ab207612, by Abcam (1 mentions)
Ab220161, by Abcam (1 mentions)
21 protocols using ab92821
1
Western Blotting for Epigenetic Regulators
2
Western Blot and 6mA Analysis
3
Comprehensive Analysis of m6A Regulators
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Ferrostatin-1 (S7243), necrostatin-1 (S8037), Z-VAD-FMK (S7023), liproxstatin-1 (S7699), sorafenib (S7397), erastin (S7242), RSL3 (S8155), MG-132 (S2619) were bought from Selleck Chemicals. CHX (C7698) and Act-D (129935) were purchased from Sigma-Aldrich. Anti-N6-methyladenosine antibody (ab208577), anti-METTL3 antibody (ab195352), anti-METTL4 (ab107540), anti-METTL14 antibody (ab220030), anti-FTO antibody (ab92821), anti-ALKBH5 antibody (ab195377), anti-YTHDF1 antibody (ab220162), anti-YTHDF2 antibody (ab220163), anti-YTHDF3 antibody (ab220161), anti-YTHDC2 antibody (ab220160), anti-HNRNPA2B1 antibody (ab31645), anti-ATG3 antibody (ab108282), anti-ATG4A antibody (ab223374), anti-ATG5 antibody (ab108327), anti-BECN1 antibody (ab207612), anti-ATG7 antibody (ab133528), anti-ATG9A antibody (ab108338), anti-ATG12 antibody (ab155589), anti-ATG16L1 antibody (ab187671), anti-LC3-I/II antibody (ab128025), anti-P62 antibody (ab109012), anti-NCOA4 antibody (ab86707), anti-FTH1 antibody (ab65080), and anti-beta actin (ab6276) antibody were purchased from Abcam Technology. Anti-WTAP antibody (sc-374280) was bought from Santa Cruz Biotechnology. Anti-Mouse IgG (G-21040) and anti-Rabbit IgG (G-21234) were bought from Thermo Fisher Scientific.
4
Protein Expression Analysis Protocol
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RIPA lysis buffer (PC101, EpiZyme, Shanghai, China) was used to extract the proteins from cells. Protein concentrations were determined using a bicinchoninic acid assay reagent kit (Beyotime, Shanghai, China). Protein lysates were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently transferred onto a polyvinylidene fluoride (PVDF, WJ002, EpiZyme) membrane. The PVDF membrane was blocked with 5% skimmed milk at 25°C for 2 h followed by overnight incubation with primary antibodies (anti-FTO, 1:1000, Abcam, USA, ab92821; anti-GAPDH, 1:1000, Abcam, USA, ab8245; anti-CTNNB1 1:1000;8480S; Cell Signaling Technology, USA, anti-slug 1:2000, Abcam, USA, ab51772; anti-ZEB1, 1:500, Abcam, USA, ab203829; anti-SOX2, 1:500, Abcam, USA, ab92494) at 4°C. The membrane was then incubated with secondary antibodies at 25°C for 2 h and binding signals were detected using an enhanced chemiluminescent kit (Beyotime, China). Afterwards, a multi-imaging system (Tanon 5200, Tanon Science & Technology Inc., Shanghai, China) was used for image acquisition and ImageJ program was used for data quantification.
5
Antibody Characterization for m6A Research
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Antibodies used in this study included a rabbit anti-m6A/m antibody (202-003, Synaptic Systems, Goettingen, Germany); a rabbit anti-human METTL3 antibody (A301-567A, Bethyl Laboratories, Inc., Montgomery, TX); a rabbit anti-human METTL14 antibody (HPA038002, Sigma, St. Louis, MO); a rabbit anti-human WTAP antibody (NBP1-83040, Novus Biologicals, LLC, Littleton, CO); a mouse anti-FTO antibody (ab92821, Abcam, Cambridge, MA); a rabbit anti-ALKBH5 antibody (HPA007196, Sigma, St. Louis, MO); a rabbit anti-human YTHDF1 antibody (ab99080, Abcam, Cambridge, MA); a rabbit anti-human YTHDF3 antibody (ab103328, Abcam, Cambridge, MA); a goat anti-mouse YTHDF3 antibody (sc-87503, Santa Cruz Inc., Dallas, TX); a rat anti-LANA antibody (ab4103, Abcam, Cambridge, MA); a rabbit anti-human YTHDF2 antibody (24744-1-AP, Proteintech Group, Rosemont, IL); a mouse anti-ORFK8 antibody (sc-57889, Santa Cruz Inc., Dallas, TX); a rabbit anti-human YTHDC1 antibody (ab133836, Abcam, Cambridge, MA); a rabbit anti-human YTHDC2 antibody (ab176846, Abcam, Cambridge, MA); a mouse anti-human β-actin antibody (sc-47778, Santa Cruz Inc., Dallas, TX). An anti-ORF57 antibody was generated by Sigma-Aldrich by immunizing a rabbit with the peptide IDGESPRFDDSIIP . The rabbit antibody to RTA and the mouse antibody to ORF65 were previously described52 (link),53 (link).
6
Western Blot Analysis of m6A Regulators
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Western blotting was performed as previously reported 22 (link)-24 (link). Briefly, total proteins were extracted from human aorta samples or HASMCs by using radioimmunoprecipitation assay (RIPA) lysis buffer. The proteins were separated by SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. After that, the membrane was blocked with 5% nonfat milk for 90 min and then incubated with the indicated primary antibody overnight at 4 °C. The primary antibodies used in this study were as follows: ALKBH5 (HPA007196, Atlas Antibodies), AIFM2/FSP1 (HPA042309, Atlas Antibodies), METTL14 (HPA038002, Atlas Antibodies), METTL3 (15073-1-AP, Proteintech), WTAP (60188-1-Ig, Proteintech), SLC3A2 (15193-1-AP, Proteintech), FTO (ab92821, Abcam), GPX4 (ab125066, Abcam), β-Actin (#8457, Cell Signaling Technology), SLC7A11 (NB300-318, Novus Biologicals), and Flag (F1804, Sigma-Aldrich) antibodies.
7
Western Blot Analysis of m6A Regulators
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Lung tissue was lysed in a RIPA buffer (Beyotime Biotechnology, China) with PMSF (Thermo Fisher, USA); and a phosphatase inhibitor cocktail (Thermo Fisher, USA) was also added to the RIPA buffer when p-eNOS was to be analyzed. The lysate was then centrifuged for 10 min at 12,000g, and the supernatant was collected. An enhanced BCA protein assay kit (Beyotime Biotechnology, China) was used to measure the protein concentration. A 10% SDS-PAGE gel was used for electrophoresis, and the electrotransfer occurred for 90 min at 110 V with 0.45 µm PVDF membranes (Merck Millipore, USA). The membranes were incubated with antibodies, and a chemiluminescence method was used for color development. The G:BOX system (Syngene, USA) was used to generate gray-scale images. Image-Pro Plus software (Media Cybernetics, USA) was used to quantify the amount of protein as gray value. The primary antibodies used in the western blots were as follows: anti-METTL3 (1:1000, ab195352, Abcam, UK), anti-METTL14 (1:2000, #51104, Cell Signaling Technology, USA), anti-WTAP (1:1000, #56501, Cell Signaling Technology), anti-FTO (1:1000, ab92821,Abcam), anti-ALKBH5 (1:500, 16837–1-AP,Proteintech, USA), anti-VEGF (1:200, 26157–1-AP,Proteintech), anti-eNOS (1:1000, ab199956,Cell Signaling Technology), anti-p-eNOS (1:1000, #9570, Cell Signaling Technology), and anti-β-actin (1:5000, A1978, Sigma-Aldrich, USA).
8
Western Blot Analysis of Brain Protein Markers
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The brain samples were lysed in RIPA lysis buffer with protease inhibitor cocktail (B14001, Bimake) by a tissue homogenizer, followed by ultrasonication and centrifugation. The bicinchoninic acid method was used to determine the concentrations of proteins that were mixed with 5 × Laemmli sample buffer and denatured for 5 min at 95 °C. A total of 30 µg of each sample was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. The membranes were incubated with blocking buffer (TBST buffer containing 5% skim milk powder) for 60 min at room temperature. Next, the membranes were incubated overnight at 4 °C with the primary antibodies. The primary antibodies were as follows: anti-FTO (1:1000, ab92821, Abcam), anti-ADRB2 (1:1000, ab182136, Abcam), anti-SIRT1 (1:1000, 8469 S, Cell Signaling Technology), anti-c-MYC (9402 S, 1:1000, Cell Signaling Technology), and anti-GAPDH (1:10000, GTX100118, GeneTex), HRP-conjugated secondary anti-rabbit (1:5000, GTX213110-01, GeneTex), and HRP-conjugated secondary anti-mouse (1:5000, GTX213111-01, GeneTex). After incubation for 1 h with the corresponding secondary antibodies, the protein bands were detected by chemiluminescence using an ECL reagent.
9
Antibody Characterization for m6A Research
10
Immunoelectron Microscopy Analysis of Cellular Proteins
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IEM analysis was performed at the electron microscopy core facility on campus. Briefly, cells were fixed for 1 h, followed by washing with 0.1 M PB buffer. Samples were then dehydrated with ethanol, embedded, cut into 80 nm-thick slides by Leice EM UC6, and then mounted on formvar/carbon-coated 200 mesh gold grids. Then slides were incubated with antibodies (p62 Sigma-Aldrich 1:20, P0067; FTO abcam, ab92821, 1:20) in 1% BSA. After rehydration with PBS and blocking with 1% BSA, slides were next washed with PBS, followed by blocking with 0.5% BSA again, and then incubated with gold-conjugated secondary antibodies (10 nm for FTO or 15 nm for p62, 1:10) in 0.5% BSA. After washing, samples were stained briefly with Uranyl Acetate and Lead Citrate, air dried, and then examined under 300KV at FEI Tecnai F30.
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