Cell lysates were immunoblotted with primary antibodies targeting ALKBH5 (Abcam), ZNF333 (Invitrogen), CDX2 (Abcam), VIL1 (Abcam), KLF4 (Abcam), YTHDF2 (Abcam), p65 (CST), p-p65 (CST), FLAG (CST), and GAPDH (Abcam). The intensity of the fluorescence was detected using a chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA), and quantified using Quantity One software (Bio-Rad, USA).
Ythdf2
Manufactured by Abcam
Sourced in United States
YTHDF2 is a protein that functions as an RNA-binding protein. It is involved in the regulation of mRNA stability and translation. YTHDF2 recognizes and binds to N6-methyladenosine (m6A) modifications in mRNA, which can lead to the degradation or repression of the modified mRNAs.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Znf333, by Thermo Fisher Scientific (1 mentions)
Alkbh5, by Abcam (1 mentions)
Chemiluminescence system, by Cytiva (1 mentions)
Gapdh, by Abcam (1 mentions)
Rbm15, by Abcam (1 mentions)
Ythdf1, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence blotting kit, by CWBIO (1 mentions)
Mettl16, by Proteintech (1 mentions)
Enhanced chemiluminescence system, by Merck Group (1 mentions)
Vinculin, by Cell Signaling Technology (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Hrp labeled secondary antibody, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
5 protocols using ythdf2
1
Immunoblotting Analysis of Cell Signaling Proteins
2
Immunohistochemical Analysis of m6A Regulators
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3
Western Blot Analysis of Protein Modifications
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The total protein was extracted using a RIPA kit (Huaxing Bio, Beijing, China), and then the protein concentration was quantified using a bicinchoninic acid (BCA) kit (Thermo Fisher). Protein samples (25 ug) were separated on electrophoresed in polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, MA, United States). After blocking in 5% skimmed milk at room temperature for 1 h, membranes were incubated with primary antibodies against FTO (Proteintech, Wuhan, China), METTL16 (Proteintech), YTHDF2 (Abcam), CBLL1 (Proteintech), and GAPDH (Huaxing bio) at 4°C overnight. The membranes were incubated with HRP-conjugated secondary antibodies (Huaxing Bio) for 1 h and visualized by an enhanced chemiluminescence blotting kit (Cwbiotech, Jiangsu, China). The intensities of the bands were quantified using Quantity One software (Bio-Rad, CA, United States). GAPDH was used as the internal control.
4
Western Blot Analysis of Signaling Proteins
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This work applied RIPA lysis buffer (Beyotime, Haimen, China) in combination with protease as well as phosphatase inhibitor cocktails (Cwbiotech, Beijing, China) for sample collection within a 30-min period on ice. Later, BCA protein assay (Beyotime) was conducted for measuring protein content. Subsequently, 8% SDS-PAGE was applied in separating 30 μg proteins. Then, transfer on PVDF membrane was performed (Millipore, Billerica, MA, USA). After being blocked by 5% BSA for an 80-min under ambient temperature for eliminating nonspecific protein binding, the membrane was subject to overnight incubation with primary antibodies (1 : 1000) under 4 °C, including YTHDF2, p53 (Abcam, Cambridge, UK), IκBα, p-IκBα, IKKα, IKKβ, p-IKKα/β, p38, p-p38, p65, p-p65, JNK, p-JNK, ERK, p-ERK, β-actin, VINCULIN, and GAPDH (Cell Signaling Technologies, Danvers, MA, USA). Membrane was rinsed TBST and further probed for a 1-h period using the 1 : 2000 diluted HRP-labeled secondary antibody (Cell Signaling Technology, Boston, MA, USA). This work employed enhanced chemiluminescence system (Millipore, Billerica, MA, USA) for protein band visualization by adopting ImageJ v1.47 software (National Institutes of Health, Bethesda, MD, USA), with GAPDH or VINCULIN being the endogenous reference.
5
Western Blot and qRT-PCR Analysis of YTHDF2 and ZC3H13
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RIPA buffer containing the protease inhibitor PMSF (Solarbio Science & Technology Company, China) was used to lyse tissues on ice, and BCA kit (Solarbio Science & Technology Company, China) was used for protein quantification. A total of 20 µg proteins were separated by 10% SDS-PAGE and electro-blotted onto nitrocellulose (NC) membrane. After sealing with skimmed milk, the NC membrane was incubated with the first antibody at 4 °C overnight. The membranes were washed and incubated with the second antibody on the shaking table at room temperature for two hours. ECL chemiluminescence kit (Advansta, USA) was used to visualize the protein bands. β-actin was used as a control. The main antibodies used in this study included YTHDF2 (1:1,000) and ZC3H13 (1:1,000) (Abcam, USA).
For mRNA quantifications of YTHDF2 and ZC3H13, cDNA was synthesized by DNase treatment and reverse transcription (TIANGEN Biotech Company, China). Real-time PCR was on TL988 Real-Time PCR Detection System (TIANLONG, China). The primers were listed inTable 1 . The mRNA levels of the selected genes were normalized to that of the reference gene β-actin, and the value were calculated by the 2−ΔΔCt method. The results are expressed as the means ± standard error based on three independent experiments.
For mRNA quantifications of YTHDF2 and ZC3H13, cDNA was synthesized by DNase treatment and reverse transcription (TIANGEN Biotech Company, China). Real-time PCR was on TL988 Real-Time PCR Detection System (TIANLONG, China). The primers were listed in
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