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Published on: January 7, 2019
Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic
Deborah L Grainger1, Lydia Kutzler2, Sharon L Rannels2
1Proteintech Group, Manchester, UK.
Abstract:
REDD1 is a transcriptional target gene of p53 and HIF-1, and an inhibitor of mTOR (mechanistic target of rapamycin) complex 1 (mTORC1)-signaling through PP2A-dependent interaction, making it an important convergence point of both tumor suppression and cell growth pathways. In accordance with this positioning, REDD1 levels are transcriptionally upregulated in response to a variety of cellular stress factors such as nutrient deprivation, hypoxia and DNA damage. In the absence of such conditions, and in particular where growth factor signaling is activated, REDD1 expression is typically negligible; therefore, it is necessary to induce REDD1 prior to experimentation or detection in model systems. Here, we evaluated the performance of a commercially available polyclonal antibody recognizing REDD1 by Western blotting in the presence of thapsigargin, a pharmacological inducer of ER stress well known to upregulate REDD1 protein expression. Further, REDD1 antibody specificity was challenged in HEK-293 cells in the presence of RNA interference and with a REDD1 (-/-) mouse embryonic fibroblast knockout cell line. Results showed reproducibility and specificity of the antibody, which was upheld in the presence of thapsigargin treatment. We conclude that this antibody can be used to reliably detect REDD1 endogenous expression in samples of both human and mouse origin.
Insights
This study validates a commercial antibody for detecting REDD1 (regulated in development and DNA damage response 1) protein. The antibody reliably detects REDD1 expression in human and mouse cells, even under stress conditions.
Area of Science:
- Molecular Biology
- Cellular Stress Response
Background:
- REDD1 (regulated in development and DNA damage response 1) is a key inhibitor of mTORC1 signaling.
- REDD1 expression is induced by cellular stress (nutrient deprivation, hypoxia, DNA damage) but typically low under growth factor signaling.
- Accurate detection of REDD1 is crucial for studying tumor suppression and cell growth pathways.
Purpose of the Study:
- To evaluate the performance and specificity of a commercially available REDD1 antibody.
- To confirm antibody reliability in detecting endogenous REDD1 under various experimental conditions.
Main Methods:
- Western blotting was used to assess antibody performance.
- Experiments involved thapsigargin treatment to induce ER stress and upregulate REDD1.
- Specificity was further tested using RNA interference and a REDD1 knockout cell line.
Main Results:
- The REDD1 antibody demonstrated reproducible and specific detection of the protein.
- Antibody performance was consistent even in the presence of thapsigargin-induced ER stress.
- Validation was successful in both human HEK-293 cells and mouse embryonic fibroblasts.
Conclusions:
- The evaluated polyclonal antibody reliably detects endogenous REDD1 expression.
- This antibody is suitable for use in both human and mouse samples.
- It can be utilized in studies involving cellular stress and mTORC1 signaling pathways.
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