Immunoprecipitation of METTL3 from 293T cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is METTL3 (D2I6O) Rabbit mAb. Western blot analysis was performed using METTL3 (D2I6O) Rabbit mAb and Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 secondary antibody.
Western blot analysis of extracts from various cell lines using METTL14 (D8K8W) Rabbit mAb.
Chromatin immunoprecipitations were performed with cross-linked chromatin from mES cells and either Virilizer (D4N8B) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Mouse Sox2 Exon 1 Primers #30180, mouse Klf2 intron 2 primers, and SimpleChIP® Mouse MYT-1 Promoter Primers #8985. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Western blot analysis of extracts from various cell lines using WTAP Antibody.
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.
Western blot analysis of extracts from various cell lines using METTL3 (D2I6O) Rabbit mAb.
Western blot analysis of extracts from various cell lines using Virilizer (D4N8B) Rabbit mAb.
|METTL3 (D2I6O) Rabbit mAb 96391||20 µl||
||H Mk M R||70||Rabbit IgG|
|METTL14 (D8K8W) Rabbit mAb 51104||20 µl||
||H Mk M R||65||Rabbit IgG|
|Virilizer (D4N8B) Rabbit mAb 88358||20 µl||
||H Mk M R||210||Rabbit IgG|
|WTAP Antibody 56501||20 µl||
||H Mk M R||55||Rabbit|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The N6-mA Methyltransferase Antibody Sampler Kit provides an economical means of detecting N6-mA methyltransfearse proteins using control antibodies against METTL3, METTL14, WTAP, and Virilizer. The kit contains enough primary antibodies to perform at least two western blot experiments.
Each antibody in the N6-mA Methyltransferase Antibody Sampler Kit detects endogenous levels of its target protein.
METTL14, METTL3, and Virilizer monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Leu297 of human METTL3 protein, Pro130 of human METTL14 protein, and Pro229 of human Virilizer protein. The WTAP polyclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human WTAP protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.
Methyltransferase-like protein 3 (METTL3) and methytransferase-like protein 14 (METTL14) are the two catalytic subunits of an N6-methyltransferase complex that methylates adenosine residues in RNA (1). Methylation of adenosine residues regulates mRNA splicing, processing, translation efficiency, editing and stability, in addition to regulating primary miRNA processing, and is critical for proper regulation of the circadian clock, embryonic stem cell self-renewal, immune tolerance, response to various stimuli, meiosis and mouse fertility (2,3). In this complex, METTL3 functions as the catalytic methyltransferase subunit and METTL14 functions as the target recognition subunit by binding to RNA (4). In addition, the Wilms tumor 1-associated protein (WTAP) functions as a regulatory subunit and is required for accumulation of the complex to nuclear speckles, which are sites of RNA processing (5). Several studies suggest a role for this complex in cancer. METTL3 expression is elevated in lung adenocarcinoma where it promotes growth, survival and invasion of human lung cancer cells (6). In addition, WTAP is over-expressed in a number of different cancers and positively regulates cell migration and invasion in glioblastoma and cholangiocarcinoma (7,8).
Virilizer is an additional protein subunit of the METTL3/METTL14 N6-methyltransferase complex and is required for methyltransferase activity (9). As part of the WTAP complex, Virilizer functions to regulate sex determination in Drosophila melanogaster through splicing of Sex-lethal (Sxl) mRNA transcripts (10,11).
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