Titrate the impact of LJH685 on Ras activation
Treat MCF10A cells (in duplicate) with dilution series of LJH685 (Stock in DMSO): 100µM, 33µM, 10µM, 3µM, 1µM, 0.3µM, 0.1µM, 0µM (control) +/- 3ng/ml EGF for 30 min. Total of 32 samples. Extract and quantify activated Ras activity levels by ELISA.
Expected results: We expect to see an increase in Ras activity as we increase the concentrations of LJH685. We should see this increase in both the presence and absence of EGF but see a much stronger effect with EGF. The 30 min sample time should be enough time to reach signaling steady state, but not long enough to impact transcriptionally controlled feedback regulators. I suspect that 10µM is enough for maximum inhibition, but this might depend on cell type.
SEEK ID: https://emsl-seek.pnnl.gov/assays/21
Experimental assay
Projects: Reverse Sensitivity Analysis for Identifying Predictive Proteomics Signa...
Investigation: hidden item
Study: hidden item
Assay position:
Assay type: Inhibitor study
Technology type: ELISA
Organisms: Homo sapiens : MCF 10A (wild-type / Epithelial)
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Created: 29th Oct 2021 at 00:51
Last updated: 8th Dec 2021 at 22:44
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I consider myself a "systems biologist", trained in a variety of different areas, but primarily interested in how organisms work as integrated, self-regulating systems. Trained in protein chemistry and cell biology, I have extensive experience in modeling biological systems as well as image analysis and other novel analytical technologies. Currently, my major focus is on integrating multiple types of 'omics data to provide insight into fundamental biological regulatory mechanisms.
A major aim of cancer systems biology is to build models that can predict the impact of these genetic disruptions to guide therapeutic interventions. A prominent driver of cancer cell growth is signaling pathway deregulation from mutations in key regulatory nodes and loss/gain in gene copy number (CNV). Recent work by our group discovered that the abundances of most signaling pathway proteins are highly conserved with signaling being controlled by only a few, low abundance key nodes. The activity ...
Public web page: https://csbconsortium.org/research-projects/reverse-sensitivity-analysis-for-identifying-predictive-proteomics-signatures-of-cancer/
Start date: 1st Apr 2019
End date: 30th Apr 2024
Genotypes: wild-type
Phenotypes: Epithelial
Comment: The MCF 10A cell line is a non-tumorigenic epithelial cell line. The cells are positive for epithelial sialomucins, cytokeratins and milk fat globule antigen. They exhibit three dimensional growth in collagen, and form domes in confluent cultures. Thus far, the cells have shown no signs of terminal differentiation or senescence. The line is responsive to insulin, glucocorticoids, cholera enterotoxin, and epidermal growth factor (EGF). By electron microscopy the cells display characteristics of luminal ductal cells but not of myoepithelial cells. They also express breast specific antigens as detected by positive reaction with MFA-Breast and MC-5 monoclonal antibodies. The calcium content of the medium exerts a strong effect on the morphology of the cells.
