Immunoblotting was carried out as previously described [44 (link)]. Antibodies against E-cadherin (3195, 1:1000), Vimentin (5741, 1:1000), p-STAT3 (9145, 1:1000), ZEB1 (70512, 1:1000) and β-actin (4970, 1:1000) were purchased from Cell Signaling Technology. Antibodies against FTO (ab126605, 1:1000), YTHDF1 (ab252563, 1:1000), YTHDF2 (ab246514, 1:1000), YTHDF3 (ab220161, 1:1000), TSG101 (ab125011, 1:1000), and CD81 (ab134045, 1:1000) were purchased from Abcam. Antibodies against IGF2BP1 (22803-1-AP, 1:1000), IGF2BP2 (11601-1-AP, 1:1000), IGF2BP3 (14642-1-AP, 1:1000) and GAPDH (60004-1-Ig, 1:2000) were purchased from Proteintech (China). Full and uncropped blots were uploaded as Supplemental Material.
Ab246514
Manufactured by Abcam
Sourced in United States
Ab246514 is a lab equipment product offered by Abcam. It is a device used for conducting scientific experiments and analyses in a laboratory setting. The core function of this product is to facilitate specific tasks required in the research and development process.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (3 mentions)
Ab220161, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ab195352, by Abcam (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab126605, by Abcam (1 mentions)
Ab134045, by Abcam (1 mentions)
Ab125011, by Abcam (1 mentions)
P stat3 9145, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ab220162, by Abcam (1 mentions)
Ab205606, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab172730, by Abcam (1 mentions)
Magna rip kit, by Merck Group (1 mentions)
Ab186733, by Abcam (1 mentions)
Ez magna rna immunoprecipitation kit, by Merck Group (1 mentions)
Streptavidin c1 magnetic beads, by Thermo Fisher Scientific (1 mentions)
11 protocols using ab246514
1
Immunoblotting of EMT and m6A Regulators
2
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).
3
YTHDF2 Immunoprecipitation and RNA Detection
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Cell lysates were obtained using the Magna RIP Kit (Millipore, Billerica, MA, USA). Immunoprecipitation was performed with 50 μL of protein A/G magnetic beads and 5 μg of anti‐YTHDF2 antibody (1:30, ab246514, Abcam), using normal rabbit IgG (1:100, ab172730, Abcam) as an NC. The captured RNA, after washing with RIP wash buffer, underwent qPCR analysis, and the data were normalized to calculate the relative expression.
4
LINC01273 RNA Interactome Profiling
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The RNA pull-down assay was conducted as previously described [23 (link)]. In brief, the biotin-labeled anti-sense DNA probes for LINC01273 were designed and synthesized by Sangon (Shanghai, China). The HCC cell lysates were then collected and incubated with LINC01273 probe at for 2 h at 4°C. RNA complexes were washed with NT2 buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl2, and 0.05% NP-40) and incubated with streptavidin-magnetic C1 beads (Cat.65001, Invitrogen) for 0.5 h at 4°C. The enriched RNA was extracted and used for qRT-PCR analysis. And RIP assay was performed using EZ Magna RNA Immunoprecipitation Kit (Cat.17–701, Millipore) according to the manufacturer’s guidelines. The IP antibody was anti-Ago2 (ab186733, Abcam) and anti-YTHDF2 (ab246514, Abcam).
5
Quantification of Protein Expression
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Proteins were extracted from cells or tissues using RIPA lysis buffer (Thermo Fisher). The protein concentration in each extract was detected using a BCA Protein Assay Kit (Beyotime). A sample of protein from each extract was separated by 10% SDS-PAGE, and the protein bands were transferred onto polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany), which were subsequently blocked with 5% BSA for 2 h. Next, the membranes were incubated for 1 h with primary antibodies against METTL3 (ab195352, 1:1000, Abcam, Cambridge, UK), YTHDF2 (ab246514, 1:1000), C3 (ab200999, 1:2000), C1q (ab11861, 1:2000), C1r (ab190800, 1:3000), C1s (ab134943, 1:2000), C1qA (ab189922, 1:2000), C1qB (ab92508, 1:2000), C1qC (ab75756, 1:1000), and GAPDH (ab8245, 1:10000, Abcam), and then incubated with a secondary antibody (Abcam) for 1 h. The immunostained protein bands were detected using an enhanced chemiluminescence reagent (Thermo Fisher).
6
Western Blot Antibody Optimization
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The following antibodies and concentrations were used: rabbit anti-GAPDH (1:1000 for Western blot; Proteintech 10494-1-AP), rabbit anti-HA (1:1000 for Western blot; CST C29F4), rabbit anti-ADARB1 (1:1000 for Wester blot; Aviva Systems OAAF02345), mouse anti-FLAG M2 (1:2000 for Western blot; Sigma F1804), rabbit anti-YTHDF1 (1:1000 for Western blot; Abcam ab252346), rabbit anti-YTHDF2 (1:2000 for Western blot; Abcam ab246514), rabbit anti-YTHDF3 (1:2000 for Western blot; Abcam ab220161), rabbit anti-RPL10 (1:1000; GeneTex GTX55169), rabbit anti-RPS3 (1:1000; CST 2579S), rabbit anti-PABP (1:2000; CST 4992S), horseradish peroxidase (HRP)-conjugated goat antirabbit (1:2500; Abcam ab6721), and HRP-conjugated goat antimouse (1:10,000; Invitrogen 62-6520).
7
Quantitative Protein Analysis via Western Blot
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The collection and quantification of total proteins from sample cells/tissues were carried out by RIPA lysis buffer (Beyotime, Shanghai, China) and BCA kits, respectively. After exposure to 10% SDS-PAGE, the separated proteins were loaded onto PVDF membranes, followed by blockage with 5% bovine serum albumin (BSA, Solarbio, Beijing, China). Then, the membranes were incubated with respective primary antibodies at 4 °C for more than 12 h. The antibodies used in this study include as follows: BDNF (1:1000, ab108319, Abcam), METTL3 (1:1000, ab195352, Abcam), and GAPDH (1:1000, ab8245, Abcam,), DGCR8 (1:1000, ab191875, Abcam), YTHDF2 (1:1000, ab246514, Abcam). On the next day, the membranes were incubated with secondary antibodies with the same origin. After being thoroughly rinsed with PBS buffer, the protein bands were visualized with the application of enhanced chemiluminescence (ECL, Millipore, USA). The original western blots were provided in Supplementary Material .
8
Western Blotting Protein Analysis Protocol
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The Western blotting assay was conducted in accordance with a previous study [24 (link)]. The cell lysate containing the protease inhibitor, phenylmethylsulfonyl fluoride (PMSF) (Beyotime, Nantong, China), was added to the cells to extract proteins and quantified using a BCA protein quantification kit (Beyotime, Nantong, China). Equal amounts of proteins were added to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the membrane was transferred, and the polyvinylidene fluoride (PVDF) membrane of the corresponding size was cut out according to the molecular weight. After blocking with 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST) for 2 h, the membranes were incubated with the corresponding primary antibodies (ASS1, 1/2000, ab170952, Abcam; YTHDF2, 1/1000, ab246514, Abcam) at 4 °C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Immunoblots were detected using an imaging system (Bio-Rad, USA). GAPDH was used as the loading control.
9
YTHDF2-Bound RNA Immunoprecipitation
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RIP assay was performed by the Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA). In brief, the harvested tumor cells were incubated with magnetic beads that coated with anti-YTHDF2 antibody (ab246514, Abcam) or isotype IgG at 4 °C overnight. Then the beads were washed six times and incubated with proteinase K for digestion. We used Phenol-chloroform-isoamyl alcohol reagent to extract the RNA in the precipitates and inputs. Reverse transcription PCR was conducted to quantify ZEB1 mRNA and the expression was normalized to control input. IgG was used to confirm the specificity of RNA–protein interactions.
10
m6A-RIP Protocol for Protein-RNA Interactions
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RIP analysis was performed with the Magna RIP RNA-binding protein immunoprecipitation kit as per the manufacturer’s protocol (Millipore, Bedford, MA). Briefly, samples were irradiated at 254 nm, 400 mJ/cm2 (Stratagene Stratalinker), followed by treatment with RIP lysis buffer. The immunoprecipitation was implemented using the with antibodies against m6A (1:800, ab208577, Abcam, Cambridge, MA), DGCR8 (1:800, ab90579, Abcam, USA), and YTHDF2 (1:1000, cat. no. ab246514, Abcam, USA) proteins. The enriched RNA was analyzed via qRT-PCR.
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