Phospho-MYOD1 (Ser200) Antibody
Product Specs
Target Background
- Using basic helix-loop-helix (bHLH) transcription factors ASCL1, ASCL2, and MYOD1, which are essential mediators of lineage specification, researchers have addressed the paradox of differential binding despite similar DNA motif preferences. While these factors exhibit distinct DNA motif preferences, this difference is not sufficient to explain the extent of their differential binding. All three factors can bind inaccessible chromatin and induce changes in chromatin accessibility and H3K27ac. PMID: 29500235
- ACL regulates the net amount of acetyl groups available, leading to alterations in acetylation of H3(K9/14) and H3(K27) at the MYOD locus, ultimately increasing MYOD expression. PMID: 29241530
- Studies have found that the MYOD transcription factor can upregulate miR-223 expression by binding to an E-box region of the gga-miR-223 gene promoter during avian myoblast differentiation. IGF2 and ZEB1 are two target genes of miR-223. PMID: 28981085
- A significant proportion, exceeding 25%, of BRAF(V600E) alleles may be associated with disease outcome in PTC patients. PMID: 27688110
- This study presents the first report of a MYOD1 (L122R) mutation in the largest cohort of 49 rhabdomyosarcomas reported so far, suggesting an association with a relatively aggressive clinical course. PMID: 27562493
- Cell transdifferentiation of primary skin fibroblasts by forced expression of the myogenic transcription factor MyoD was achieved through quantitative analyses of gene expression and chromatin accessibility profiles. PMID: 28977539
- Analysis of the chromatin status of the Cdkn1c promoter and KvDMR1 in unresponsive compared to responsive cell types revealed that their differential responsiveness to MyoD-dependent induction of the gene is not solely determined by methylation status, but rather involves differential H3 lysine 9 dimethylation at KvDMR1. PMID: 27611768
- Data suggest that MeCP2 promotes gastric cancer (GC) cell proliferation via FOXF1-mediated Wnt5a/beta-Catenin signaling pathway, and suppresses GC cell apoptosis through MYOD1-mediated Caspase-3 signaling pathway. PMID: 28131747
- Findings on Pax7 and MyoD protein expression suggest that proliferation and differentiation of skeletal muscle stem cells are affected in ALS patients, and the myogenic processes cannot overcome the denervation-induced wasting. PMID: 27195289
- The molecular pathogenesis of radiotherapy-induced muscle fibrosis involves the TGF-beta1 pathway and its repression of MyoD expression. Results indicate a correlation between traditional swallow therapy/neuromuscular electrical stimulation combined therapy and the restoration of TGF-beta1/MyoD homeostasis in cervical muscles. PMID: 27144672
- Unmethylated MYOD1 gene is associated with chemoradiation resistance in Invasive Cervical Carcinoma. PMID: 26344356
- This study provides the first description of a human phenotype seemingly resulting from a MYOD1 mutation. The presentation with Lethal fetal akinesia deformation sequence is consistent with a substantial body of research demonstrating that in mice, MyoD is a major controller of precursor cell commitment to the myogenic differentiation program. PMID: 26733463
- These results suggest that sarcoma metastasis can be partially controlled through Pax7/MyoD-dependent activation of miR-182, providing insight into the role of myogenic transcription factors in sarcoma progression. PMID: 26234681
- The mechanism of bakuchiol-induced myogenesis is described. PMID: 26902638
- These observations demonstrate, for the first time, that Wnt3a can directly activate MyoD expression by targeting cis-elements in the DE and the L fragment. PMID: 25651906
- Studies indicate that MyoD occupies multiple promoters that induce the transcription of genes essential for establishing the myogenic fate, and is also implicated as a mediator of many chromatin modifying enzymes for their recruitment to myogenic enhancers. PMID: 24905980
- MUNC is not a classic cis-acting enhancer RNA (e-RNA) acting exclusively by stimulating the neighboring MyoD gene. PMID: 25403490
- MyoD acts to promote SC proliferation and transition of cells into differentiation, while myogenin is known to drive terminal differentiation. PMID: 25108351
- A recurring point mutation in MYOD1 is found in 10% of embryonal rhabdomyosarcomas with distinct clinical features and poor prognosis. PMID: 25002625
- Spindle cell and sclerosing rhabdomyosarcoma exhibit recurrent MYOD1 mutations, consistent with a single pathological entity, regardless of age at presentation. PMID: 24824843
- MyoD was required for the induction of FGF21 gene transcription by mitochondrial dysfunction. PMID: 25055037
- Analysis of a mutation in MYOD1 suggests a potential role in the progression of embryonal rhabdomyosarcoma and may be associated with mutations altering PI3K-AKT pathway components. PMID: 24793135
- MYOD1 homozygous mutations are frequent, recurrent, and pathognomonic events in adult-type spindle cell Rhabdomyosarcoma. PMID: 24272621
- While expression of MyoD in a proliferating tumor is insufficient to prevent tumor progression, its expression in the cerebellum hinders medulloblastoma genesis. PMID: 24092238
- Direct reprogramming of fibroblasts to myocytes was achieved via bacterial injection of MyoD protein. PMID: 23438194
- MYOD1-transduced amnion-derived cells are capable of expressing dystrophin, which is necessary for myogenic differentiation. PMID: 22727434
- CRABP2 promotes myoblast differentiation and is modulated by the transcription factors MyoD and Sp1 in C2C12 cells. PMID: 23383201
- SREBP-1 regulates muscle protein synthesis through the downregulation of the expression of MYOD1, MYOG, and MEF2C factors. PMID: 23226416
- Using both primary human muscle cells and RD rhabdomyosarcoma cells, studies have shown that MyoD binds in a similar genome-wide pattern in both tumor and normal cells but binds poorly at a subset of myogenic genes that fail to activate in the tumor cells. PMID: 23230269
- Promoter gene hypermethylation of the MYOD-1 gene increases significantly with age in normal individuals and thus may offer potential as a putative biomarker for colorectal cancer. PMID: 22591756
- Human squamous cell carcinomas and malignant melanomas contain significantly more Myo/Nog cells than basal cell carcinomas. PMID: 22621191
- Results suggest that MyoD and TIP120B potentiate each other at gene expression and post-translation levels, respectively, which may promote myogenesis cooperatively. PMID: 22613845
- The involvement of HUWE1 in the ubiquitination and proteasomal degradation of MyoD was described. PMID: 22277673
- BAF60c-MyoD complex directs recruitment of SWI/SNF to muscle loci in response to differentiation cues. PMID: 22068056
- Data demonstrate a radical acceleration of iPSC creation with a fusion gene between Oct4 and the powerful transactivation domain (TAD) of MyoD. PMID: 21732495
- Using MYOD1, this study shows that a nucleosome-depleted region at the minimal enhancer region allows reprogramming to be initiated, which occurs in response to signals such as the forced expression of Myod1 in fibroblasts. PMID: 22153073
- Myofibroblasts demonstrate the capacity for de-differentiation and proliferation by modulation of endogenous levels of MyoD. PMID: 21440539
- MyoD can play an active role in Alveolar rhabdomyosarcoma (ARMS) by augmenting Pax7-FKHR function. PMID: 21321994
- Increases in MYOD indicate that 1 week of conventional resistance exercise may increase myogenic activity, including satellite cell proliferation and differentiation, in younger men. PMID: 21326383
- Mef2d, Six4, and p38alpha MAPK function coordinately as regulators of a master regulator to mediate expression of MyoD target genes. PMID: 20716948
- Knockdown of MyoD and PEA3 attenuated MDR1 expression and increased the sensitivity of multidrug resistant cancer cells to cytotoxic drugs that were transported by P-gp in SGC7901/VCR cells. PMID: 20980337
- Transgenic Pax7 and MyoD are not essential for myogenic differentiation and participation of bone marrow-derived myogenic progenitors in muscle regeneration. PMID: 20333749
- Calpain 3 participates in the establishment of the pool of reserve cells by decreasing the transcriptional activity of the key myogenic regulator MyoD via proteolysis, independently of the ubiquitin-proteasome degradation pathway. PMID: 20139084
- MYOD1 hypermethylation plays a significant role in colorectal cancer and may serve as a novel prognostic factor. PMID: 14767572
- MyoD modulates the rate of Id1 degradation, suggesting a dynamic interplay between these factors. PMID: 15163661
- Hypermethylation of MYOD1 is statistically significantly associated with poor disease outcome in cervical cancer. PMID: 15251938
- MYOD1 degradation is modulated by E12 and E47. PMID: 16007194
- This review highlights studies of molecular mechanisms by which the muscle-specific myogenic basic helix-loop-helix protein MyoD interacts with other regulatory factors to coordinate gene expression in a controlled and ordered manner. PMID: 16099183
- The expression of MyoD1 was more sensitive but less specific in patients with rhabdomyosarcoma. PMID: 16435141
- Results establish that cdk9/cyclin T2a-mediated coactivation of MyoD depends on serine 37 phosphorylation. PMID: 16841087
Q&A
Basic Research Questions
-
What is MYOD1 and why is the phosphorylation at Serine 200 significant?
MYOD1 (Myoblast Determination Protein 1) is a nuclear protein belonging to the basic helix-loop-helix family of transcription factors and the myogenic factors subfamily. It regulates muscle cell differentiation by inducing cell cycle arrest required for myogenic initiation and plays a crucial role in muscle regeneration . Phosphorylation at Serine 200 serves as a regulatory switch, with high phosphorylation observed in proliferating myoblasts and substantial dephosphorylation occurring during differentiation . This phosphorylation state directly impacts MYOD1's transcriptional activity and its role in muscle development.
-
What are the key characteristics of Phospho-MYOD1 (Ser200) antibodies?
Phospho-MYOD1 (Ser200) antibodies are typically rabbit polyclonal antibodies that specifically detect endogenous levels of MYOD1 only when phosphorylated at serine 200 . These antibodies recognize the peptide sequence containing phosphorylated Ser200 (A-S-S(p)-P-R) derived from human MYOD1 . They demonstrate reactivity across human, mouse, and rat samples, making them versatile tools for comparative studies across these mammalian species . These antibodies are affinity-purified, with non-phosphopeptide reactive antibodies removed by chromatography to ensure specificity .
-
What applications are Phospho-MYOD1 (Ser200) antibodies suitable for?
Phospho-MYOD1 (Ser200) antibodies are primarily validated for Western Blot (WB) applications, where they typically detect a band of approximately 40kDa . Depending on the specific product, these antibodies may also be suitable for Immunohistochemistry (IHC), Immunofluorescence (IF), and ELISA applications . For Western Blot applications, the recommended dilution range is typically 1:500-1:1,000, though optimal dilutions should be determined empirically for each specific application .
Advanced Research Questions
-
How is MYOD1 Ser200 phosphorylation regulated during the cell cycle?
MYOD1 undergoes cell cycle-dependent phosphorylation at Ser200, primarily mediated by cyclin-dependent kinases. Research has demonstrated that MYOD1 can be efficiently phosphorylated in vitro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts . Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis has revealed that cdk1 and cdk2 specifically phosphorylate MyoD on serine 200 in proliferative myoblasts . This phosphorylation mechanism helps coordinate MYOD1 activity with cell cycle progression during myogenesis.
-
What is the relationship between MYOD1 Ser200 phosphorylation and p38-γ signaling?
Research utilizing phospho-Ser200-specific antibodies has revealed a relationship between p38-γ signaling and MYOD1 phosphorylation. Specifically, p38-γ-dependent gene silencing appears to restrict entry into the myogenic differentiation program, with p38-γ potentially regulating MYOD1 phosphorylation at Ser200 . While the complete signaling pathway remains under investigation, this interaction suggests a complex regulatory network controlling MYOD1 activity during muscle cell fate determination.
-
How does MYOD1 function in circadian rhythm regulation and how might Ser200 phosphorylation affect this role?
MYOD1 functions as a clock amplifier and critical co-factor in the molecular clock machinery. It works with core clock factors to regulate the expression of clock-controlled genes in skeletal muscle . MYOD1 can transcriptionally activate and enhance the amplitude of the Bmal1 promoter through interaction with a non-canonical E-box motif (5'-CAGGGA-3') located downstream of the Bmal1 transcription start site . While the direct impact of Ser200 phosphorylation on this function hasn't been fully characterized, the phosphorylation state likely modulates MYOD1's interactions with clock components and its ability to enhance circadian amplitude.
-
What experimental approaches can distinguish between the effects of MYOD1 Ser200 phosphorylation versus other regulatory mechanisms?
To isolate the specific effects of Ser200 phosphorylation, researchers should employ a multi-faceted approach including: (1) Site-directed mutagenesis to create phospho-mimetic (S200D/E) or phospho-deficient (S200A) MYOD1 variants for functional studies ; (2) Temporal correlation of Ser200 phosphorylation with biological events using phospho-specific antibodies; (3) Pharmacological manipulation with specific kinase inhibitors; (4) Genetic approaches targeting the kinases responsible for Ser200 phosphorylation; and (5) Comparative analysis with other MYOD1 post-translational modifications to establish hierarchical relationships.
Methodological Considerations
-
What controls should be included when using Phospho-MYOD1 (Ser200) antibodies?
For rigorous validation of Phospho-MYOD1 (Ser200) antibody results, researchers should include: (1) Total MYOD1 antibody detection in parallel to assess phosphorylation relative to total protein levels; (2) Phosphatase-treated samples as negative controls; (3) Samples from different physiological states (proliferating vs. differentiating myoblasts) as biological controls ; (4) Peptide competition assays using both phosphorylated and non-phosphorylated peptides; and (5) When possible, genetically modified samples (MYOD1 knockout or Ser200 mutants) to confirm specificity.
-
What are the optimal sample preparation methods for preserving MYOD1 Ser200 phosphorylation?
To maintain the phosphorylation status of MYOD1 at Ser200, samples should be: (1) Processed rapidly after collection; (2) Lysed in buffers containing phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate); (3) Maintained at cold temperatures throughout processing; (4) Prepared with protease inhibitors to prevent degradation; and (5) Stored appropriately (-80°C for long-term storage). Additionally, avoid multiple freeze-thaw cycles as they can affect phosphorylation status.
-
How can phosphorylation-specific tryptic peptide mapping be used to confirm Phospho-MYOD1 (Ser200) antibody specificity?
Two-dimensional tryptic phosphopeptide mapping is a powerful approach for confirming phosphorylation site specificity. This method involves: (1) Immunoprecipitating 32P-labeled MYOD1 from cells or phosphorylating bacterially expressed MYOD1 in vitro with purified kinases; (2) Digesting the protein with trypsin; (3) Separating peptides by two-dimensional electrophoresis (first dimension at pH 1.9, second dimension in phosphochromo buffer); (4) Comparing wild-type MYOD1 with site-specific mutants (e.g., MYOD-Ala200) . This approach provides definitive confirmation of the phosphorylation site recognized by the antibody.
-
What approaches can be used to study the dynamics of MYOD1 Ser200 phosphorylation during myogenesis?
To effectively investigate Ser200 phosphorylation dynamics, researchers should consider: (1) Time-course experiments with samples collected at defined intervals during myoblast differentiation; (2) Parallel analysis of cell cycle markers, differentiation markers, and MYOD1 Ser200 phosphorylation; (3) Synchronized cell populations to normalize for cell cycle effects; (4) Quantitative Western blotting with both phospho-specific and total MYOD1 antibodies; and (5) Live-cell imaging approaches using phospho-sensor technologies when available.
-
How can researchers integrate Phospho-MYOD1 (Ser200) data with genomic and transcriptomic analyses?
To correlate MYOD1 phosphorylation status with its genomic functions, researchers should consider: (1) Combining ChIP-seq using Phospho-MYOD1 (Ser200) antibodies with RNA-seq to link phosphorylation state to target gene expression; (2) Comparing binding profiles of wild-type and phospho-mutant MYOD1 to identify phosphorylation-dependent target genes; (3) Integrating phosphorylation data with time-course transcriptomics during myogenesis; (4) Analyzing co-factor recruitment to MYOD1 binding sites in relation to phosphorylation status; and (5) Employing systems biology approaches to model the impact of Ser200 phosphorylation on the broader myogenic program.
