Western blot analysis of extracts from Daudi, EL4, and Raji cells, untreated (-) or treated with MG-132 #2194 (10 μM, 18hr; +), using Phospho-c-Myc (Thr58) (E4Z2K) Rabbit mAb (upper), c-Myc (E5Q6W) Rabbit mAb #18583 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower). Note that Raji cells have a point mutation at Thr58.
Western blot analysis of extracts from HDLM-2 cells, untreated (-) or treated with MG-132 #2194 (10 μM, 18 hr; +) or GSK-3 Inhibitor XXII, Compound A (5 μM, 18hr; +), using Phospho-c-Myc (Thr58) (E4Z2K) Rabbit mAb (upper), c-Myc (E5Q6W) Rabbit mAb #18583 (middle) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
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.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Phospho-c-Myc (Thr58) (E4Z2K) Rabbit mAb recognizes endogenous levels of c-Myc protein only when phosphorylated at Thr58. This antibody may react to c-Myc when dually phosphorylated at Thr58 and Ser62. This phosphorylation site is also conserved at Thr58 in N-Myc.
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr58 of human c-Myc protein.
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).
Explore pathways + proteins related to this product.
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