Protein extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel, transferred to polyvinylidene fluoride membranes, and probed with antibodies against ATM (2873S, Cell Signaling Technology, CST); p-ATM -S1981 (AP0008, ABclonal); METTL3 (15073-1-AP, Proteintech); YTHDF1 (17479-1-AP, Proteintech); YTHDF2 (24744-1-AP, Proteintech); YTHDF3 (25537-1-AP, Proteintech); FTO (TA809392, OriGene); m6A (202003, Synaptic Systems); BRCA1 (20649-1-AP, Proteintech); H2A.X (A11361, ABclonal); γH2A.X (AP0687, ABclonal ); GAPDH(60004-1-Ig, Proteintech); eIF3A (3411T, CST); Flag (F1804, Sigma-Aldrich); anti‐Rabbit IgG HRP‐linked antibody (7074; CST); Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (SA00001-1, Proteintech); α-Tubulin (66031-1-Ig, Proteintech); CHK1(bs-1681R, Bioss); CHK2 (252092, Zenbio); p-CHK2-S19(AP0862, ABclonal); Anti-HA tag monoclonal antibody (TA100012, OriGene); Mouse-IgG (BA1046, Boster); Rabbit IgG (B900610, Proteintech); p21(10355-1-AP, Proteintech); 53BP1 (BA2878, Boster); CDK1 (D160158, BBI life sciences); CDK2 (D199431, BBI life sciences); Cyclin B2 (21644-1-AP, Proteintech).
Ythdf3
Manufactured by Proteintech
YTHDF3 is a protein that functions as an RNA-binding protein, specifically recognizing and binding to N6-methyladenosine (m6A) modifications in RNA. It is involved in the regulation of gene expression and mRNA metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Ythdf2, by Proteintech (3 mentions)
Ythdf1, by Proteintech (3 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Mettl3, by Proteintech (2 mentions)
Gapdh, by Proteintech (2 mentions)
Peroxidase conjugated affinipure goat anti mouse igg h l, by Proteintech (1 mentions)
P21 10355 1 ap, by Proteintech (1 mentions)
Cyclin b2, by Proteintech (1 mentions)
Brca1, by Proteintech (1 mentions)
Gapdh 60004 1 ig, by Proteintech (1 mentions)
α tubulin, by Proteintech (1 mentions)
Rabbit igg, by Proteintech (1 mentions)
0.45 μm polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Anti ha c29f4 rabbit mab, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Mettl14, by Proteintech (1 mentions)
Rbms1, by Abcam (1 mentions)
Vinculin, by Proteintech (1 mentions)
3 protocols using ythdf3
1
Western Blot Analysis of DNA Damage Markers
2
Western Blot for Protein Expression
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Cells were washed twice with phosphate-buffered saline (PBS) and lysed with RIPA Lysis Buffer (Beyotime, #P0013C) supplemented with phenylmethanesulfonyl fluoride (Sigma-Aldrich, #p7626) and cOmplete™ (Roche, #4693132001) on ice. Supernatant of cell lysates was collected after centrifugation and denatured at 100°C with loading buffer. Samples were loaded onto an 8% 15-well sodium dodecyl sulphate-polyacrylamide gelelectrophoresis gel, and the gel was transferred to a 0.45-μm polyvinylidene difluoride membrane (Merckmillipore, #IPVH00010) after electrophoresis. Membranes were blocked with 5% non-fat powdered milk (Sangon Biotech, #A600669-0250) and incubated overnight at 4° cold room with anti-HA (C29F4) rabbit mAb (Cell Signaling Technology, #3724), GAPDH (Proteintech, #60004-1-Ig), YTHDF1 (Proteintech, #17479-1-AP), YTHDF2 (Proteintech, #24744-1-AP) and YTHDF3 (Proteintech, #25537-1-AP) antibodies in TBST (TBS + 0.5% Tween-20) with 1% bovine serum albumin (BSA, Sangon Biotech, # A600332-0100). After washing three times with TBST, membranes were incubated with secondary antibodies (Cell Signaling Technology, #7074 and #7076) for 1 h at RT. The membrane was washed and incubated with an enhanced chemiluminescence ECL (Thermo scientific, #34580) for 2 min then imaged by ChemiDoc XRS+ System (BIO RAD, #1708265).
3
Western Blotting of m6A Regulators
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Cells were washed twice with cold PBS. Then the cells were lysed with RIPA lysis buffer (50 × 10−3m Tris‐HCl (pH 7.4), 150 × 10−3m NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin) for 30 min. The cell lysate was centrifuged at 12 000 rpm for 15 min. The protein concentration was measured using BCA protein assay kit. Equal amounts of total protein were separated on 10% or 15% SDS‐PAGE and transferred onto NC membranes, blocked with 5% nonfat milk at room temperature for 1 h, and incubated with primary antibodies at 4 ˚C overnight. After washed three times, the membranes were incubated with secondary Ig conjugated HRP for 1 h at room temperature. Protein bands were visualized with the ECL enhanced chemiluminescence regent Kit (NCM Biotech). The following antibodies were used: RBMS1 (Abcam ab150353), S100P (Proteintech 11803‐1‐AP), YTHDF1 (Proteintech 17479‐1‐AP), YTHDF2 (Proteintech 24744‐1‐AP), YTHDF3 (Proteintech 25537‐1‐AP), METTL3 (Proteintech 15073‐1‐AP), METTL14 (Proteintech 26158‐1‐AP), FTO (Proteintech 27226‐1‐AP), m6A (Proteintech 68055‐1‐lg), FLAG (Sigma 1804), V5 (Proteintech 14440‐1‐AP), VINCULIN (Proteintech 66305‐1‐Ig), GAPDH (Proteintech 60004‐1‐Ig).
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