Revision 4

#71283

Store at -20C

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

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

Applications:
W, IP, IF-F, IF-IC, eCLIP

Reactivity:
H M R Mk

Sensitivity:
Endogenous

MW (kDa):
65

Source/Isotype:
Rabbit IgG

UniProt ID:
#Q9Y5A9

Entrez-Gene Id:
51441

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
Immunofluorescence (Frozen) 1:50 - 1:200
Immunofluorescence (Immunocytochemistry) 1:800 - 1:3200
eCLIP 1:200
For more information about the RBP-eCLIP service please visit Eclipsebio.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity/Sensitivity

YTHDF2 (E2I2H) Rabbit mAb recognizes endogenous levels of total YTHDF2 protein. Experiments utilizing overexpression constructs indicated that this antibody does not cross-react with other YTHDF proteins.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly167 of human YTHDF2 protein.

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 members of the YT521B homology (YTH) domain-containing family (YTHDF), consisting of YTHDF1, YTHDF2, and YTHDF3. All three members of the YTHDF family are primarily cytosolic proteins that share similar sequence and domain structure, including a conserved C-terminal YTH domain that specifically interacts with m6A (1). 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 (2). Conversely, YTHDF2 binding appears to promote mRNA degradation, but has minimal effect on translation efficiency (3). 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 (4). Additional studies offer a different viewpoint, suggesting that all three YTHDF proteins initiate mRNA degradation (5), or mediate increased mRNA stability and protein expression (6), promoting the idea that these proteins may carry out similar rather than distinct functions.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

W: Western Blotting IP: Immunoprecipitation IF-F: Immunofluorescence (Frozen) IF-IC: Immunofluorescence (Immunocytochemistry) eCLIP: eCLIP

Cross-Reactivity Key

H: Human M: Mouse R: Rat Mk: Monkey

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

Alexa Fluor is a registered trademark of Life Technologies Corporation.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 4

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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).
Western Blotting Image 1: YTHDF2 (E2I2H) Rabbit mAb
Western blot analysis of extracts from various cell lines using YTHDF2 (E2I2H) Rabbit mAb.
Western Blotting Image 2: YTHDF2 (E2I2H) Rabbit mAb
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.
Immunoprecipitation Image 1: YTHDF2 (E2I2H) Rabbit mAb
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 4

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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).
Immunofluorescence Image 1: YTHDF2 (E2I2H) Rabbit mAb
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).
Immunofluorescence Image 2: YTHDF2 (E2I2H) Rabbit mAb
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).
Immunofluorescence Image 3: YTHDF2 (E2I2H) Rabbit mAb
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 4

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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).
Immunofluorescence Image 1: YTHDF2 (E2I2H) Rabbit mAb
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).
Immunofluorescence Image 2: YTHDF2 (E2I2H) Rabbit mAb
Enhanced cross-linking and immunoprecipitation (eCLIP) was performed with RNA from K-562 cells and YTHDF2 (E2I2H) Rabbit mAb using a protocol based on the RBP-eCLIP Kit from EclipseBio. The figure shows binding across the OXA1L transcript. Data is kindly provided by the laboratory of Dr. Gene Yeo and used with permission.
eCLIP Image 1: YTHDF2 (E2I2H) Rabbit mAb
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.