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80523
N6-Methyladenosine (m6A) Binding Protein Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

N6-Methyladenosine (m6A) Binding Protein Antibody Sampler Kit #80523

Citations (0)
Immunoprecipitation of YTHDF3 protein from 293T cell extracts. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is YTHDF3 Antibody. Western blot analysis was performed using YTHDF3 Antibody. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
Western blot analysis of extracts from 293T cells, transfected with control siRNA (-) or YTHDF3 siRNA (+), using YTHDF3 Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunoprecipitation of YTHDC2 protein from HeLa cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is YTHDC2 (E6H7U) Rabbit mAb. Western blot analysis was performed using YTHDC2 (E6H7U) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
Western blot analysis of extracts from 293T cells, either transfected with non-targeting siRNA (293T siNT) or siRNA targeting YTHDC2 (293T siYTHDC2), and various other cell lines using YTHDC2 (E6H7U) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
Immunoprecipitation of YTHDF1 protein from MCF7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is YTHDF1 (E9P6V) Rabbit mAb. Western blot analysis was performed using YTHDF1 (E9P6V) Rabbit mAb. Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) #93702 was used as the secondary antibody.
Western blot analysis of extracts from HCT 116 cells, transfected with control siRNA (-) or YTHDF1 siRNA (+), using YTHDF1 (E9P6V) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Confocal immunofluorescent analysis of HCT 116 cells, mock transfected (left, high-expressing) or transfected with YTHDF1 siRNA (right, low-expressing), using YTHDF1 (E9P6V) Rabbit mAb (green) and DAPI #4083 (blue).
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Immunoprecipitation of YTHDF2 protein from 293T cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is YTHDF2 (E2I2H) Rabbit mAb. Western blot analysis was performed using YTHDF2 (E2I2H) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as the secondary antibody.
Western blot analysis of extracts from 293T cells, either transfected with non-targeting siRNA (siNT) or siRNA targeting YTHDF2 (siYTHDF2), and various cell lines using YTHDF2 (E2I2H) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Confocal immunofluorescent analysis of HCT 116 cells mock transfected (left, high-expressing) or transfected with siRNA directed against human YTHDF2 (right, low-expressing) using YTHDF2 (E2I2H) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Confocal immunofluorescent analysis of mouse hippocampus using YTHDF2 (E2I2H) Rabbit mAb (red) and GFAP (GA5) Mouse mAb (Alexa Fluor® 488 Conjugate) #3655 (green). Actin filaments were labeled with DyLight 650 Phalloidin #12956 (cyan pseudocolor). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Confocal immunofluorescent analysis of mouse medulla oblongata using YTHDF2 (E2I2H) Rabbit mAb (red) and GFAP (GA5) Mouse mAb (Alexa Fluor® 488 Conjugate) #3655 (green). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Confocal immunofluorescent analysis of mouse retina using YTHDF2 (E2I2H) Rabbit mAb (red) and GFAP (GA5) Mouse mAb (Alexa Fluor® 488 Conjugate) #3655 (green). Actin filaments were labeled with DyLight 650 Phalloidin #12956 (cyan pseudocolor). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Confocal Immunofluorescent analysis of U-2 OS cells untreated (left) or treated with sodium arsenite (500 µM, 1 hr) (right) using YTHDF2 (E2I2H) Rabbit mAb (green) and G3BP1 (TT-Y) Mouse mAb (red). After blocking free secondary antibody binding sites with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, the cells were then labeled using GM130 (D6B1) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #59890 (cyan pseudocolor). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Western blot analysis of extracts from various cell lines using YTHDC1 (E4I9E) Rabbit mAb.
Western blot analysis of extracts from various cell lines using YTHDF3 Antibody.
Western blot analysis of extracts from various cell lines and tissues using YTHDC2 (E6H7U) Rabbit mAb.
Western blot analysis of extracts from various cell lines using YTHDF1 (E9P6V) Rabbit mAb.
Confocal immunofluorescent analysis of U-2 OS cells, untreated (left) or treated with sodium arsenite (right; 500 µM, 1 hr), using YTHDF1 (E9P6V) Rabbit mAb (green), DyLight 650 Phalloidin #12956 (red), and DAPI #4083 (blue).
Immunoprecipitation of YTHDC1 from HCT 116 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is YTHDC1 (E4I9E) Antibody. Western blot analysis was performed using YTHDC1 (E4I9E) Rabbit mAb.
Western blot analysis of extracts from various cell lines using YTHDF2 (E2I2H) Rabbit mAb.
To Purchase # 80523
Cat. # Size Qty. Price
80523T
1 Kit  (5 x 20 microliters)

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
YTHDF1 (E9P6V) Rabbit mAb 57530 20 µl
  • WB
  • IP
  • IF
H M R Mk 70 Rabbit IgG
YTHDF2 (E2I2H) Rabbit mAb 71283 20 µl
  • WB
  • IP
  • IF
H M R Mk 65 Rabbit IgG
YTHDF3 Antibody 24206 20 µl
  • WB
  • IP
H M R Mk 70 Rabbit 
YTHDC1 (E4I9E) Rabbit mAb 77422 20 µl
  • WB
  • IP
H M R Mk 110 Rabbit IgG
YTHDC2 (E6H7U) Rabbit mAb 46324 20 µl
  • WB
  • IP
H M R Mk 160 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Rab Goat 

Product Description

The N6-Methyladenosine (m6A) Binding Protein Antibody Sampler Kit provides an economical means of evaluating total levels of YTH domain-containing proteins. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Each antibody in the N6-Methyladenosine (m6A) Binding Protein Antibody Sampler Kit detects endogenous levels of its target protein. Experiments utilizing overexpression constructs indicated that YTHDF3 Antibody may weakly cross-react with YTHDF1.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly90 of human YTHDF1 protein, Gly167 of human YTHDF2 protein, Phe557 of human YTHDC1 protein, and Gly1243 of human YTHDC2 protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly169 of human YTHDF3 protein, and are purified by peptide affinity chromatography.

Background

N6-methyladenosine (m6A) is an abundant RNA modification that plays an important role in mRNA splicing, processing, and stability. The m6A modification is specifically recognized by YT521B homology (YTH) domain-containing proteins, consisting of five members in mammals: YTH domain-containing proteins 1 and 2 (YTHDC1 and YTHDC2), and YTH domain-containing family proteins 1, 2, and 3 (YTHDF1, YTHDF2, and YTHDF3) (1). YTHDC1, also known as splicing factor YT521, regulates alternative splicing by functioning as a key regulator of exon-inclusion or exon-skipping. YTHDC1 promotes exon-inclusion by recruiting pre-mRNA splicing factor SRSF3 to regions containing m6A, while repressing exon-skipping by blocking SRSF10 binding to these same regions (2). Increased expression of YTHDC1 promotes malignant endometrial carcinoma (EC) through alternative splicing of vascular endothelial growth factor-A (VEGF-A), resulting in an increase in VEGF-165 isoform and increased EC cell invasion (3). YTHDC2 functions to enhance the translation efficiency of target mRNAs and may play a role in spermatogenesis (4). All three members of the YTHDF family are paralogs that share similar sequence and domain structure, including the conserved C-terminal YTH domain that specifically interacts with m6A (5). Despite these similarities, recent studies suggest that YTHDF proteins are involved in distinct regulatory functions with minimal overlap. Specifically, YTHDF1 binding has been reported to promote enhanced mRNA translation, but has no measurable effect on mRNA stability (6). Conversely, YTHDF2 binding appears to promote mRNA degradation, but has minimal effect on translation efficiency (7). The function of YTHDF3 is less clear, but it has been proposed to function as an auxiliary protein for both YTHDF1 and YTHDF2, helping to promote either increased mRNA translation or decay, respectively (8). Additional studies offer a different viewpoint, suggesting that all three YTHDF proteins initiate mRNA degradation, or mediate increased mRNA stability and protein expression, promoting the idea that these proteins may carry out similar rather than distinct functions (9,10).

Limited Uses

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For Research Use Only. Not for Use in Diagnostic Procedures.
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