#57868 DNMT3B (E8A8A) XP® Rabbit mAb
|DNMT3B (E8A8A) XP(R) recognizes endogenous levels of total DNMT3B protein. This antibody does not cross-react with DNMT3A or DNMT1.|
|Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human DNMT3B protein.|
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Western blot analysis of extracts from various cell lines using DNMT3B (E8A8A) XP® Rabbit mAb.
Immunoprecipitation of DNMT3B from NCCIT cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is DNMT3B (E8A8A) XP® Rabbit mAb. Western blot analysis was performed using DNMT3B (E8A8A) XP® Rabbit mAb and Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127.
Confocal immunofluorescent analysis of NCCIT cells (left, high-expressing), HCT 116 DNMT3B wild-type cells (middle, low-expressing), and HCT 116 DNMT3B knockout cells (right, negative) using DNMT3B (E8A8A) Rabbit XP® mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Flow cytometric analysis of NCCIT cells (green), HCT116 WT cells (red) or HCT116 DNMT3B KO cells (blue), using DNMT3A (E8A8A) XP® Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either DNMT3B (E8A8A) XP® Rabbit mAb #57868 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® Human EIF4G1 Intron 8 Primers #29118, human CROCC intron 1 primers, and SimpleChIP® Human GAPDH Promoter Primers #4471. 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.
Methylation of DNA at cytosine residues in mammalian cells is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting and development (1,2). Three families of mammalian DNA methyltransferases have been identified: DNMT1, DNMT2 and DNMT3 (1,2). DNMT1 is constitutively expressed in proliferating cells and functions as a maintenance methyltransferase, transferring proper methylation patterns to newly synthesized DNA during replication. DNMT3A and DNMT3B are strongly expressed in embryonic stem cells with reduced expression in adult somatic tissues. DNMT3A and DNMT3B function as de novo methyltransferases that methylate previously unmethylated regions of DNA. DNMT2 is expressed at low levels in adult somatic tissues and its inactivation affects neither de novo nor maintenance DNA methylation. DNMT1, DNMT3A and DNMT3B together form a protein complex that interacts with histone deacetylases (HDAC1, HDAC2, Sin3A), transcriptional repressor proteins (RB, TAZ-1) and heterochromatin proteins (HP1, SUV39H1), to maintain proper levels of DNA methylation and facilitate gene silencing (3-8). Improper DNA methylation contributes to diseased states such as cancer (1,2). Hypermethylation of promoter CpG islands within tumor suppressor genes correlates with gene silencing and the development of cancer. In addition, hypomethylation of bulk genomic DNA correlates with and may contribute to the onset of cancer. DNMT1, DNMT3A and DNMT3B are over-expressed in many cancers, including acute and chronic myelogenous leukemias, in addition to colon, breast and stomach carcinomas (9-12).
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Tween is a registered trademark of ICI Americas, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
ProLong is a registered trademark of Life Technologies Corporation.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.