Total RNA immunoprecipitation was performed with the EZ-Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (Merk) following the manufacturer’s instructions. Immunoprecipitation were carried out using 5 μg of anti-ADAR1 antibody (Bethyl), anti-YTHDF1 (Abcam, Ab220162 and Ab99080), anti-FLAG (SIGMA), or normal rabbit/mouse immunoglobulin G (IgG) as control and incubating at 4 °C overnight. Precipitated RNA was then centrifuged at 15,000g for 30′ at 4 °C and pellet resuspended in 15 μl of RNase-free water.
Ab220162
Manufactured by Abcam
Sourced in United States
Ab220162 is a primary antibody that can be used for the detection of target proteins in various applications such as Western blotting, immunohistochemistry, and flow cytometry. The specific function and details of the antibody are not provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab195352, by Abcam (5 mentions)
Ab220163, by Abcam (4 mentions)
Ab220161, by Abcam (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca assay kit, by Beyotime (2 mentions)
Ab97051, by Abcam (2 mentions)
Ab16663, by Abcam (2 mentions)
Ecl solution, by Thermo Fisher Scientific (2 mentions)
Ab2735, by Abcam (2 mentions)
Ab104836, by Abcam (2 mentions)
Ab99080, by Abcam (1 mentions)
Ab133666, by Abcam (1 mentions)
Ab220161, by Proteintech (1 mentions)
Ab181602, by Proteintech (1 mentions)
Ab108596, by Abcam (1 mentions)
Ab76302, by Abcam (1 mentions)
Ab76315, by Abcam (1 mentions)
18 protocols using ab220162
1
Immunoprecipitation of RNA-Binding Proteins
2
Nuclear and Cytoplasmic Protein Extraction
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The cytoplasm and nuclear fraction of cells were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, China), according to the manufacturer’s instructions. Whole cell lysates or the nuclear/cytoplasm fractions were subjected to SDS-PAGE and immunoblotting, as described in our previously published study [24 (link), 25 (link)]. Primary antibodies against STAT3 (Abcam, ab119352), phosphorylated STAT3 (p-STAT3) (Abcam, ab76315), GAPDH (Abcam, ab181602), GNAS (Proteintech, 10,150–2-AP), YTHDF1 (Abcam, ab220162), YTHDF2 (Abcam, ab220163), YTHDF3 (Abcam, ab220161), P65 (Proteintech, 10,745–1-AP), phosphorylated P65 (pp65) (Abcam, ab76302), JAK1 (Abcam, ab133666), and JAK2 (Abcam, ab108596) were used.
3
RIP-qRT-PCR Assay for m6A Readers
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RIP assays were performed essentially as described in our previously published study [24 (link), 25 (link)]. In brief, cells were lysed using polysome lysis buffer (5 mM HEPES (pH 7.4), 85 mM KCl, 1 mM DTT, 5 mM PMSF, 0.5% NP40, supplemented with RNase inhibitors (Invitrogen, USA) and PIC (protease inhibitors cocktail, Roche, Switzerland)) on ice for 10 min. After centrifugation, the supernatant was collected with 10% of the lysate serving as “input”. The remainder of the lysate was incubated with 50 μl of protein A/G magnetic beads (Life Technologies, USA) coupled with 2 μg of primary antibodies rotated overnight at 4 °C with IgG antibody as the control. RNA was isolated using TRIzol (Invitrogen, USA) and reverse-transcribed into cDNA for qRT-PCR detection using a Takara SYBR green kit (Takara, Japan). Primary antibodies against YTHDF1 (Abcam, ab220162), YTHDF2 (Abcam, ab220163), YTHDF3 (Abcam, ab220161), and N6-methyladenosine (m6A) (Abcam, ab220161) were used.
4
Analyzing YTHDF Proteins in Cardiomyocytes
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Proteins were extracted from heart tissues or isolated mice primary cardiomyocytes with RIPA Lysis Buffer (Beyotime, China), and protein concentration was determined using the BCA protein assay kit (Beyotime, China). Protein samples were separated on a 10% SDS-PAGE gel and then transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: YTHDF1 (ab220162, Abcam, USA), YTHDF2 (ab246514, Abcam, USA), YTHDF3 (ab220161, Abcam, USA), ANP (ab262703, Abcam, USA), flag (ab205606, Abcam, USA), and GAPDH (ab8245, Abcam, USA), followed by incubation with secondary antibodies for 1 h. The protein blots were visualized using the ECL kit (Pierce, USA).
5
Western Blot Analysis of Apoptosis and RNA Modification
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RIPA buffer (Beyotime) was used to extract the total cellular proteins and a BCA assay kit (Beyotime) was used to determine the protein concentration in each extract. A 40 μg sample of protein from each extract was separated by 10% SDS-PAGE and the protein bands were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA), which were subsequently blocked with 5% non-fat milk in TBST for 2 h at room temperature. Next, the membranes were incubated with primary antibodies against caspase-3 (ab32042, Abcam, Cambridge MA, USA), Bax (ab182734, Abcam), Bcl-2 (ab182858, Abcam), PARP1 (ab227244, Abcam), BRCA1 (ab131360, Abcam), BRCA2 (ab239375, Abcam), YTHDF1 (ab220162, Abcam), WTAP (ab232392, Abcam), ALKBH5 (ABE547, Sigma-Aldrich), and β-actin (ab8227, Abcam) at 4 °C overnight, followed by a 1.5 h incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature. Finally, the target proteins were visualized with an enhanced chemiluminescence kit (Pierce, Waltham, MA, USA).
6
Characterizing RNA-Protein Interactions
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The interaction within RNA binding proteins and mRNA was identified using RIP-PCR. In brief, the RIP experiment was carried out by EZ-Magna RIP Kit (Millipore) according to the manufacturer’s protocol. ASMCs were lysed in complete RIP lysis buffer, and the cell extract was incubated with protein A/G agarose beads conjugated by anti-YTHDF1 (ab220162, 1:30; Abcam) or control IgG (ab172730; Abcam) for 2 h at 4 °C. After being washed, beads were incubated with Proteinase K to remove protein in complex. Lastly, the purified RNAs were subjected to qRT-PCR analysis.
7
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.
8
Quantification of m6A Regulators via Western Blot
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Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China), supplementing with protease inhibitor. Protein concentration was detected using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, MA, USA). The lysed protein extract was subjected to 10% SDS-PAGE and was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) and then blocked with 5% nonfat milk. Membranes were incubated with the primary antibodies (Abcam, Cambridge, MA, USA) overnight at 4°C and with anti-METTL3 (Abcam; ab195352, 1:1,000), anti-YTHDF1 antibody (Abcam; ab220162, 1:1,000), and anti-c-Myc antibody (Abcam; ab56, 1:1,000). Then, the members were conjugated to horseradish peroxidase (HRP) for 2 h at room temperature as controls. Finally, the blots were detected by using enhanced chemiluminescence (ECL) detection reagent (Thermo Fisher Scientific).
9
Western Blot Analysis of YTHDF1 and CONT7
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Western blotting was performed as previously described [23 (link)]. After digestion and suspension, the transfected MG63 and HOS cells were added into a 6-well plate at the cell density of 2 × 106/well; and the protein expression was measured after culturing for 24 h. The protein lysis buffer (RIPA: PMSF = 100:1) was used to extract total proteins, while the BCA method was used to measure total proteins. About 60 µg protein from each group was subjected to electrophoresis and then transferred into the PVDF membrane. Subsequently, the membrane was blocked at room temperature for 1 h and incubated with the primary antibody at 4°C for overnight. The following day, horseradish peroxidase labeled secondary antibody was added. Color development ensued following addition of chromogenic solution; photographs were taken for analysis 1 h post incubation. The antibodies used were as follows: YTHDF1 (ab220162, 1/1000, abcam), CONT7 (ab195587, 1/1000, abcam), GAPDH (60,004-1-Ig, 1/1000, proteintech), and the secondary antibodies peroxidase-conjugated with the anti-rabbit and anti-mouse (Santa Cruz Biotechnology).
10
Protein Extraction and Analysis of RNA Methyltransferases
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Protein extraction was performed using protein extraction kit (Key Gene, KGP9100) form CC cells. Lipid proteins were appended into gels and subjected nitrocellulose membranes. Total proteins were harvested as indicated in figure legends, separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nitrocellulose membrane was incubated with antibodies against METTL3 (ab195352, Abcam, 1:1000), against YTHDF1 (ab220162), against HK2 (ab209847, Abcam, 1:1000). GAPDH was used as normalized control.
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