Determination of Copy Number of c-Myc Protein per Cell by Quantitative Western Blotting

Rudolph, Claudia; Adam, Gerold; Simm, Andreas

doi:10.1006/abio.1999.3095

Cited by 48 publications

(37 citation statements)

References 41 publications

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“…Extracts were prepared from transduced REF52 and Rat-1 cells using the freeze-thaw method of Rudolph et al (1999). Extracted proteins were resolved by SDS-PAGE, and detected by Western blotting using the anti-HAM antibody (Herbst and Tansey, 2000).…”

Section: Determination Of Steady-state Protein Levelsmentioning

confidence: 99%

Multiple cell-type-specific elements regulate Myc protein stability

et al. 2004

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Myc is a highly unstable transcription factor that is destroyed by ubiquitin (Ub)-mediated proteolysis. We have previously identified an amino-terminal 'degron' within Myc that signals its destruction; this degron spans the transcriptional activation domain of Myc, and includes two highly conserved regions called Myc boxes I and II. We now report the identification of a second element -the D-element -which is also required for Myc proteolysis. The centrally located D-element is distinct from the PEST domain in Myc, but includes Myc box III, a third highly conserved region with no previously known function. We show that deletion of the D-element stabilizes the Myc protein without affecting its ubiquitylation, and report that the D-element and the degron act in a cell-typespecific manner to direct Myc proteolysis. These data thus demonstrate that Myc stability is regulated at both the ubiquitylation and postubiquitylation levels, and reveal that substrates of the Ub -proteasome system can be targeted for destruction differently in different cell types.

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Section: Determination Of Steady-state Protein Levelsmentioning

confidence: 99%

Multiple cell-type-specific elements regulate Myc protein stability

et al. 2004

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“…For the initial conditions of miRs, we compared literature data and assumed that miR levels are on the order of 10 3 to 10 4 copies per cell in normoxia; the concentrations (in microMolar) used as initial conditions in the model are computed using 1 pL cell compartment volumes based on literature measurements [125–128]. Absolute levels of the different proteins in the model are estimated to be on the order of 10 4 to 10 6 copies per cell based on experimental measurements of several pathway-related proteins including Myc, p53 and calmodulin [115, 129, 130]. …”

Section: Methodsmentioning

confidence: 99%

Transcriptional and Post-Transcriptional Regulation of Thrombospondin-1 Expression: A Computational Model

2017

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Hypoxia is an important physiological stress signal that drives angiogenesis, the formation of new blood vessels. Besides an increase in the production of pro-angiogenic signals such as vascular endothelial growth factor (VEGF), hypoxia also stimulates the production of anti-angiogenic signals. Thrombospondin-1 (TSP-1) is one of the anti-angiogenic factors whose synthesis is driven by hypoxia. Cellular synthesis of TSP-1 is tightly regulated by different intermediate biomolecules including proteins that interact with hypoxia-inducible factors (HIFs), transcription factors that are activated by receptor and intracellular signaling, and microRNAs which are small non-coding RNA molecules that function in post-transcriptional modification of gene expression. Here we present a computational model that describes the mechanistic interactions between intracellular biomolecules and cooperation between signaling pathways that together make up the complex network of TSP-1 regulation both at the transcriptional and post-transcriptional level. Assisted by the model, we conduct in silico experiments to compare the efficacy of different therapeutic strategies designed to modulate TSP-1 synthesis in conditions that simulate tumor and peripheral arterial disease microenvironment. We conclude that TSP-1 production in endothelial cells depends on not only the availability of certain growth factors but also the fine-tuned signaling cascades that are initiated by hypoxia.

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“…Total cellular protein was extracted from Urp-treated smooth muscle cells by using an alternate freeze-thaw protocol as previously described (Rudolf et al, 1999). Briefly, media was removed and the cells were washed with ice cold PBS.…”

Section: Western Blot Analysismentioning

confidence: 99%

Transcriptional profiling of the megabladder mouse: A unique model of bladder dysmorphogenesis

Singh¹,

Robinson²,

Ismail³

et al. 2007

Developmental Dynamics

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Recent studies in our lab identified a mutant mouse model of obstructive nephropathy designated mgb for megabladder. Homozygotic mgb mice (mgb؊/؊) develop lower urinary tract obstruction in utero due to a lack of bladder smooth muscle differentiation. This defect is the result of a random transgene insertion/translocation into chromosomes 11 and 16. Transcriptional profiling identified a significantly over-expressed cluster of gene products located on the translocated fragment of chromosome 16 including urotensin II-related peptide (Urp), which was shown to be preferentially over-expressed in developing mgb؊/؊ bladders. Pathway analysis of mgb microarray data indicated dysregulation of at least 60 gene products associated with smooth muscle development. In conclusion, the results of this study indicate that the molecular pathways controlling normal smooth muscle development are severely altered in mgb؊/؊ bladders, and provide the first evidence that Urp may play a critical role in bladder smooth muscle development. Developmental Dynamics 237:170 -186, 2008.

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Determination of Copy Number of c-Myc Protein per Cell by Quantitative Western Blotting

Cited by 48 publications

References 41 publications

Multiple cell-type-specific elements regulate Myc protein stability

Multiple cell-type-specific elements regulate Myc protein stability

Transcriptional and Post-Transcriptional Regulation of Thrombospondin-1 Expression: A Computational Model

Transcriptional profiling of the megabladder mouse: A unique model of bladder dysmorphogenesis

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