BersinBio RNA pull-down kit (BersinBio, Guangzhou, China) was used to performed RNA pulldown. Targeted IL-18 biotin labelled probes were designed and synthesized by GenePharma (Shanghai, China). RNA-protein complexes were formed by incubating specific probes with cell lysates. Then, the complex was separated by streptavidin conjugated magnetic beads. After separation by 10% polyacrylamide gel electrophoresis (PAGE), Western blot was performed to measure YTHDF2 (#80014, Cell Signaling) level.
Ythdf2
Manufactured by Cell Signaling Technology
Sourced in Morocco
YTHDF2 is a protein involved in the regulation of RNA metabolism. It is a member of the YTH domain family, which are readers of the N6-methyladenosine (m6A) RNA modification. YTHDF2 binds to m6A-modified RNAs and facilitates their recruitment to processing bodies for degradation.
Automatically generated - may contain errors
Lab products found in correlation
Mettl3, by Cell Signaling Technology (2 mentions)
Rna pull down kit, by BersinBio (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Inhibitor cocktail, by Roche (1 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ubiquitin, by Cell Signaling Technology (1 mentions)
Abcg2, by Cell Signaling Technology (1 mentions)
Keap1, by Santa Cruz Biotechnology (1 mentions)
P irf7, by Cell Signaling Technology (1 mentions)
P stat1, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
P irf3, by Cell Signaling Technology (1 mentions)
Mettl14, by Cell Signaling Technology (1 mentions)
Flag tag (flag), by Cell Signaling Technology (1 mentions)
Stat1, by Cell Signaling Technology (1 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (1 mentions)
6 protocols using ythdf2
1
RNA Pulldown Assay for YTHDF2
2
Protein Expression Analysis of Signaling Pathways
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After lysis of the tissues in precooled radioimmunoprecipitation assay buffer, 40 µg of protein were electrophoresed and transferred to a membrane. Next, the antibody was incubated at 4°C overnight. Finally, a chemiluminescent substrate was used for imaging, and the gray scale of the protein band was photographed for quantitative analysis to determine the relative protein expression levels. Cell Signaling Technology (Danvers, MA) supplied the primary antibodies used in this study, including YTHDF2, Wnt, β-Catenin, GSK3β, and all the secondary antibodies. Proteintech (Wuhan, China) provided primary antibodies against α-SMA, collagenI, METTL3, METTL14 ALKBH5, FTO, WTAP, and NOVA2.
3
Western Blot Analysis of METTL3 and YTHDF2
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Cells were ruptured with RIPA buffer (Beyotime) containing cocktail inhibitor (Roche). Cell lysates were resolved by SDS-PAGE and transferred onto PVDF membranes (Millipore). The membranes were blocked and then incubated with primary antibodies overnight at 4 °C. Specific antibodies used are listed below: METTL3 (Cell Signaling Technology), YTHDF2 (Cell Signaling Technology) and GAPDH (Proteintech). Subsequently, the membranes were incubated with corresponding secondary antibodies and detected by ECL Western Blotting Substrate (Thermo).
4
Western Blot Analysis of Protein Interactions
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Total cell extracts were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). Western blot was performed as previously described [17 (link)]. Primary antibodies against KEAP1 (1:500, sc-514,914, USA) and ARIH1 (1:500, sc-514,551) were purchased from Santa Cruz (MD, USA). Primary antibodies against Cleaved-caspase-3 (#9664), caspase-3 (#9662), PARP (#9542), cleaved-PARP (#9541), vimentin (#5741), ubiquitin (#8240), Flag (#14,793), HA(#3724), GFP (#2555), FTO (#31,687), METTL3 (#86,132), METTL14 (#51,104), YTHDF2 (#80,014), STAT1 (#9172),p-STAT1 (#8826), P65 (#8242), p-p65 (#3033), RIG-I (#3743), IRF7 (#4920), IRF3 (#11,904), p-IRF7 (#24,129), p-IRF3 (#4947), PCNA (#2586), SOX2 (#23,064), ABCG2 (#4477), NAONG (#4903), GAPDH (#2118), and β-actin (#4967) were purchased from Cell Signaling Technology. Densitometry analysis of Western blots was performed using Image J and the band densities were normalized to GAPDH. The control was set to 1 for plotted graphs. Protein identification was performed by LC-MS/MS. Briefly, 50 µg proteins was subjected to SDS-PAGE and the silver stained protein bands were excised and analyzed by LC-MS/MS (BGI, Shenzhen, China.
5
Western Blot Analysis of Protein Expression
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Total protein was extracted from cell lines and lysed via RIPA buffer. Degenerated protein concentration was measured by bichinchoninic acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). In each experiment, 20 μg protein samples were separated in 10% or 15% SDS-PAGE gel and transferred onto nitrocellulose membrane. After 5% non-fat milk blocking, the blots were incubated with primary antibodies including YTHDF2 (1:1000 dilution, #71283, Cell signaling), HNRNPA2B1 (1:2000 dilution, #9304, Cell signaling), HNRNPC (1:1000 dilution, HPA051075, Sigma) and internal control α-Tubulin (1:2000 dilution, #3873, Cell signaling).
6
Antibody Validation for Stemness Markers
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Anti-CD133 (catalog no. 130-090-853) and PE conjugated Anti-CD133 antibodies were purchased from Miltenyi Biotec. YTHDF2 (catalog no. 80014), SOX2 (catalog no. 23064S), AXIN2 (catalog no. 20540-1-AP) and c-MYC (catalog no. 2276S) antibodies were from Cell Signaling Technology. Anti-E2F3 (catalog no. 27615-1-AP) and IGF2BP2 (catalog no. 11601-1-AP) antibodies were from Proteintech. Anti-digoxin (catalog no. ab51949) antibody was obtained from Abcam. β-actin (catalog no. RM2001) antibody was purchased from Beijing Ray antibody Biotech. ZIC2 (catalog no. ARP35821_P050) antibody was purchased from Aviva Systems Biology. Alexa-594-, Alexa-488-and Alexa-647-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Invitrogen. HRP-conjugated secondary antibody was purchased from Sungene Biotech. Polymer HRP and AP detection kits were from GBI labs. Biotin labeld RNA mix (catalog no. 11685597910) was from Rhoche. Chemiluminescent nucleic acid detection module (catalog no. 89880) was from Thermo Scientific. ChIP assay kit (catalog no. 17-295) was from Miltenyi Biotec. Supplements N2 and B27 were purchased from Life Technologies.
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