Western blot analysis of extracts from KARPAS-299, cells untreated (-) or treated with MG-132 #2194 (10 μM, overnight; +) using Phospho-c-Myc (Ser62) (E1J4K) Rabbit mAb. The phospho-specificity of the antibody was demonstrated by peptide competition using phosphopeptides containing Ser62, Thr58/Ser62, Thr58, or a nonphosphorylated peptide as indicated.
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT and either c-Myc (E5Q6W) 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 human ATF-4 Promoter Primers, SimpleChIP® Human NPM1 Intron 1 Primers #4779, and SimpleChIP® Human α Satellite Repeat Primers #4486. 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 KARPAS-299 and HCT 116 cells, untreated (-) or treated with MG-132 #2194 (10 μM, overnight; +), using Phospho-c-Myc (Ser62) (E1J4K) Rabbit mAb (upper), c-Myc (D84C12) XP® Rabbit mAb #5605 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Flow cytometric analysis of IMR-32 cells (blue, negative) and RPMI 8226 cells (green, positive) using c-Myc (E5Q6W) Rabbit mAb (solid lines) or a 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.
Confocal immunofluorescent analysis of SCLC-21H cells (left, positive) and IMR-32 cells (right, negative) using c-Myc (E5Q6W) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red).
Immunoprecipitation of c-Myc from KG-1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is c-Myc (E5Q6W) Rabbit mAb. Western blot was performed using c-Myc (E5Q6W) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
Western blot analysis of extracts from various cell lines using c-Myc (E5Q6W) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
PhosphoPlus® Duets from Cell Signaling Technology (CST) provide a means to assess protein activation status. Each Duet contains an activation-state and total protein antibody to your target of interest. These antibodies have been selected from CST's product offering based upon superior performance in specified applications.
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).
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