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Printable Handouts
Navigable Slide Index
- Introduction
- Cellular signalling and pathway deconvolution
- Applying RNAi to the experimental process
- GeneBLAzer: beta-lactamase technology
- Beta lactamase: ratiometric assay and RNAi
- Why study EGFR pathway?
- RNAi and kinase inhibitor target qualification
- Experimental process - RNAi
- ME180AP-1 EGF titration
- RNAi in mammalian cells
- Stealth RNAi: increased stability
- RNAi analysis - qPCR
- Functional cellular RNAi time-course
- Target qualification at the protein level
- EGFR stimulation-controls - immunocytochemistry
- Kinetics of EGFR autophosphorylation
- EGFR RNAi knockdown on a cellular imaging level
- Classic experimental process - kinase inhibitors
- Small molecule inhibitor panel
- Inhibitor panel - results
- Dose response of EGFr inhibitors
- Cellular phosphorylation assay
- Cellular phosphorylation assay - dose response
- Cellular phosphorylation assay - specificity
- Kinase inhibitor structures
- Combining RNAi and kinase inhibitors
- RNAi + kinase inhibitor
- RNAi + KI (MEK1) in EGFR pathway
- RNAi + KI "cocktail" synergistic potential
- RNAi + KI (EGFR) in EGFR pathway
- RNAi + KI effects - same target
- Conclusions
Topics Covered
- Use of functional cell-based reporter system (Cell Sensor(tm) technology) with RNAi
- From target knockdown to compound screening and verification in biochemical assays
- Analysis of small-molecule protein kinase inhibitors
- Epidermal growth factor receptor (EGFR) signalling pathway
- StealthTM RNAi, second-generation RNAi compounds
- Integration of a cell-based assay system, RNAi and biochemical assay technologies
- Kinase discovery platform to identify biologically relevant kinase targets
- Characterization of kinase inhibitors in systems
- Possible synergistic effects of combining RNAi and kinase inhibitors
Talk Citation
O'Grady, M. (2015, November 13). Cell signalling pathways and RNAi [Video file]. In The Biomedical & Life Sciences Collection, Henry Stewart Talks. Retrieved November 21, 2024, from https://doi.org/10.69645/PPTR4703.Export Citation (RIS)
Publication History
Financial Disclosures
- Mr. Michael O'Grady has not informed HSTalks of any commercial/financial relationship that it is appropriate to disclose.
A selection of talks on Cell Biology
Transcript
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0:00
Cellular signaling pathways and RNAi. Michael O'Grady.
0:06
Cellular signaling and signal
transduction pathways and they're linked to many diverse disease processes,
such as Cancer, Arthritis,
and even in development has led to a plethora of studies in labs
throughout the world to deconvolve these essential biological pathways.
The advent of RNAi much like PCR to molecular cloning 15 to 20 years ago,
has led to a resurgence in a deconvolution
of these major pathways in biological processes.
This slide serves to show the complicated branches involved in
one particular pathway which will be studied in this particular presentation.
This is the EGF receptor pathway.
As can be seen in this arrow upon stimulation with epidermal growth factor ligand,
this can lead on one branch to the activation of the Janus Kinase or JAK1,
which then leads to
signal transduction phosphorylation of the STAT3 transcription factor,
which is then translocated into the nucleus binding to its response element SIE,
leading to transcriptional events which multiply
its effects seen at the level of the receptor.
Another branch goes through the adapter proteins Shk and SOS-1 into
the multibranch kinases involved in activating the AP-1 response element,
which is the classic Jun/Fos dimer transcription factor binding down to
this element which has been shown to definitely be involved in the neoplastic process.
This slide serves as a background for applying RNAi to