Activation or inhibition of signaling pathways can be employed to test hypotheses that a particular pathway is involved in your experimental model. We’ll look at how to combine modulators with selection of antibody targets and controls in western blot experiments.
Explore signaling pathway diagrams: cst-science.com/dv3n4o
Activators & Inhibitors available from CST: cst-science.com/diubr9
Contents:
0:00 Introduction
0:35 Example: detection of phosphorylated EGFR
1:24 Stripping and re-probing blots
1:46 Phosphatase controls
2:41 Combining treatments and antibody readouts
3:30 Final thoughts
Transcript:
Characterizing the activity of signaling pathways is a common research objective in the study of cancer or other diseases. Today, we’ll focus on modulation of signaling pathways using activators and inhibitors, with western blot as a readout. Many of the experimental design principles can be applied beyond western blot, as well.
As an example, consider the EGF receptor tyrosine kinase, EGFR, which autophosphorylates and activates itself after EGF ligand binding, and can activate multiple downstream pathways. To investigate which specific sites on your target are phosphorylated, select a phospho-specific antibody validated for western blot.
This blot shows an antibody that detects EGFR only when phosphorylated at tyrosine 1173. Input samples were cell lysates from either control cells, cells activated with EGF, or EGF-activated cells pre-treated with gefitinib, an inhibitor of EGFR kinase. The absence of a band in the last lane suggests that EGFR phosphorylation at tyrosine 1173 was inhibited, but further controls are required.
For western blots using phospho specific antibodies, use Stripping Buffer to remove antibodies from the membrane, and then re-probe it with an antibody that recognizes total protein. This important control is used to confirm protein degradation or changes in expression are not occurring, and supports the interpretation of the phospho-EGFR blot.
During validation of phospho-specific antibodies at CST, we also compare untreated lysates to lysates treated with phosphatase. You may decide to include a phosphatase control to re-confirm antibody phospho-specificity for your experimental model. For all other samples, adding phosphatase and protease inhibitors to the lysis buffer is recommended to avoid loss of target phosphorylation and total protein level during sample handling.
Activated surface receptors may turn on one or more pathways, depending on the biological context. By selecting antibodies for intracellular signaling proteins, such as AKT, you can investigate which pathways are responding. Reading the literature, and checking pathway diagrams from cellsignal dot com slash pathways, can give you an idea of the logical relationships between proteins in a pathway, and help you generate hypotheses for your experiment.
Once you’ve identified the pathway or pathway branch you want to investigate, you can select targets. One approach is to treat your sample with an inhibitor that targets an upstream node, and read out with one or more antibodies specific for downstream targets in different branches or pathways.
For example, phosphorylation of the EML4-ALK fusion oncoprotein is reduced by the kinase inhibitor crizotinib. By running parallel western blot experiments, using both phospho and total antibodies for ERK and AKT, we can observe reduced phosphorylation of these proteins as a result of EML4-ALK inhibition. The interpretation is that activation of ERK and AKT signaling pathways are dependent on EML4-ALK activation in this model.
These are just some of the approaches you can use as a starting point for designing your experiments. When selecting targets and antibodies, this is also the time to think about what controls you’ll need to properly interpret your data.
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17 июл 2024
