Dataset Information

Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts.

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.

SUBMITTER: Grainger DL

PROVIDER: S-EPMC4893971 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts.

Grainger Deborah L DL Kutzler Lydia L Rannels Sharon L SL Kimball Scot R SR

F1000Research 20160301

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, a ...[more]

PMID: 27335637

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Project description:Coccidioidomycosis, also called valley fever (VF), is a fungal infection with endemicity in desert regions of the western United States as well as certain arid regions of Central and South America. Laboratory-based diagnosis of VF often relies on the composite results from three serologic-based diagnostics, complement fixation, immunodiffusion, and enzyme immunoassay (EIA). EIA is commonly performed in clinical laboratories because results can be obtained in a few hours. Two commercially available EIAs, IMMY clarus Coccidioides antibody and Meridian Premier Coccidioides, look for the presence of anticoccidioidal IgG and IgM in patient sera that are diluted 1:441. Per regulatory requirements, this dilution step must be verified with a dilution step control despite not being provided as a reagent in either FDA-approved EIA kit. Therefore, clinical laboratories collect and reuse patient sera in subsequent tests that had a positive result in a previous test. This is a nonstandard process, reinforcing the need for a consistent and reliable dilution control. Here, we evaluate the performance of a humanized IgG and IgM antibody as a dilution control in both EIA kits. Both humanized IgG and IgM work well in each EIA and meet the appropriate threshold for positivity. IMPORTANCE In southwestern and western regions of the United States, at least half a million diagnostic tests for coccidioidomycosis (valley fever) are run annually. Enzyme immunoassays (EIAs) are blood tests which require precise dilution of patient serum prior to testing. To ensure patient serum is properly diluted, there is a regulatory requirement to ensure the dilution step is accurate. Two FDA-approved EIAs used to aid in the diagnosis of coccidioidomycosis do not contain controls for this dilution step, leaving clinical laboratories with the only option of using previously positive patient sera, which may not react in a reliable or predictable manner. Here, we evaluate a humanized monoclonal antibody against a coccidioidal antigen and its utility as a dilution control in both available commercial EIAs. The use of a humanized monoclonal antibody provides a standardized and well-characterized dilution control for use in serological assays that aid in diagnosis of coccidioidomycosis.

Dataset Information

Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts.

Publications

Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts.

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