Revision 3

#70365

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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, W-S, IP

Reactivity:
H M R

Sensitivity:
Endogenous

MW (kDa):
62

Source/Isotype:
Rabbit IgG

UniProt ID:
#P01106

Entrez-Gene Id:
4609

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:50 - 1:250
Immunoprecipitation 1:200

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

Phospho-c-Myc (Thr244) (F3G5K) Rabbit Monoclonal Antibody recognizes endogenous levels of c-Myc protein only when phosphorylated at Thr244. Thr244 is equivalent to Thr259 in canonical isoform 2 of c-Myc protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr244 of human c-Myc protein.

Background

Members of the Myc/Max/Mad network function as transcriptional regulators with roles in various aspects of cell behavior, including proliferation, differentiation, and apoptosis (1). These proteins share a common basic-helix-loop-helix leucine zipper (bHLH-ZIP) motif required for dimerization and DNA-binding. Max was originally discovered based on its ability to associate with c-Myc and found to be required for the ability of Myc to bind DNA and activate transcription (2). Subsequently, Max has been viewed as a central component of the transcriptional network, forming homodimers as well as heterodimers with other members of the Myc and Mad families (1). The association between Max and either Myc or Mad can have opposing effects on transcriptional regulation and cell behavior (1). The Mad family consists of four related proteins; Mad1, Mad2 (Mxi1), Mad3, and Mad4, and the more distantly related members of the bHLH-ZIP family, Mnt and Mga. Like Myc, the Mad proteins are tightly regulated with short half-lives. In general, Mad family members interfere with Myc-mediated processes, such as proliferation, transformation, and prevention of apoptosis by inhibiting transcription (3,4).

Phosphorylation of c-Myc at Thr58 and Ser62 can control proteasomal-dependent degradation of the transcription factor. Phosphorylation of c-Myc at these sites is a stepwise process whereby mitogens, mitosis, or cellular stress induce phosphorylation at Ser62, which serves as a priming site for GSK-3 phosphorylation of Thr58 (5-9). A second phosphodegron was identified at Thr244/Thr248 that acts cooperatively with Thr58/Ser62 to promote c-Myc ubiquitination and proteasomal degradation (10).

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 W-S: Simple Western™ IP: Immunoprecipitation

Cross-Reactivity Key

H: Human M: Mouse R: Rat

Trademarks and Patents

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

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 3

Cell Signaling Technology Logo
Western blot analysis of extracts from Raji cells, untreated (-) or treated with MG-132 #2194 (10 μM, 18 hr; +), and untreated (-) IMR-32 cells using Phospho-c-Myc (Thr244) (F3G5K) Rabbit mAb (upper), c-Myc (E5Q6W) Rabbit mAb #18583 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Negative expression of c-Myc protein in IMR-32 cells is consistent with the predicted expression pattern.
Western Blotting Image 1: Phospho-c-Myc (Thr244) (F3G5K) Rabbit Monoclonal Antibody
Western blot analysis of extracts from Daudi cells, untreated (-) or treated with MG-132 #2194 (10 μM, 18 hr; +), using Phospho-c-Myc (Thr244) (F3G5K) Rabbit mAb (upper), c-Myc (E5Q6W) Rabbit mAb #18583 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western Blotting Image 2: Phospho-c-Myc (Thr244) (F3G5K) Rabbit Monoclonal Antibody
Western blot analysis of extracts from MEF cells, untreated (-) or treated with MG-132 #2194 (25 μM, 6 hr; +) and RBL-2H3 cells, untreated (-) or treated with MG-132 #2194 (10 μM, 18 hr; +), using Phospho-c-Myc (Thr244) (F3G5K) Rabbit mAb (upper), c-Myc (E5Q6W) Rabbit mAb #18583 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western Blotting Image 3: Phospho-c-Myc (Thr244) (F3G5K) Rabbit Monoclonal Antibody
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 3

Cell Signaling Technology Logo
Western blot analysis of extracts from Daudi cells, untreated (-) or treated with MG-132 #2194 (10 μM, 18 hr; +), using Phospho-c-Myc (Thr244) (F3G5K) Rabbit mAb. The phospho-specificity of the antibody was demonstrated by peptide competition using a phosphopeptide containing Thr244 or a nonphosphorylated peptide, as indicated.
Western Blotting Image 4: Phospho-c-Myc (Thr244) (F3G5K) Rabbit Monoclonal Antibody
Simple WesternTM analysis of lysates (0.1 mg/mL) from Daudi + MG-132 using Phospho-c-Myc (Thr244) (F3G5K) Rabbit mAb #70365. The virtual lane view (left) shows the target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the JessTM Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Western Blotting Image 1: Phospho-c-Myc (Thr244) (F3G5K) Rabbit Monoclonal Antibody
Immunoprecipitation of phospho-c-Myc (Thr244) protein from Daudi cell extracts. Cells were treated with MG-132 #2194 (10 μM, 18 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-c-Myc (Thr244) (F3G5K) Rabbit mAb. Western blot analysis was performed using Phospho-c-Myc (Thr244) (F3G5K) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
Immunoprecipitation Image 1: Phospho-c-Myc (Thr244) (F3G5K) Rabbit Monoclonal Antibody
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.