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.
Highlights
Mechanistic target of rapamycin complex 1 is a central signaling node in the cellular response to nutrient availability and growth factor signaling, initiating and regulating processes such as protein synthesis, ribosome biogenesis and de novo lipogenesis when active
REDD1 known as DDIT4 (DNA damage-inducible transcript 4 protein) or RTP801, is a 232 amino-acid upstream repressor of Mechanistic target of rapamycin complex 1 (mTORC1) activity[3,4,5] that is transcriptionally upregulated by growth factor signaling and in response to amino acid deprivation, among other stimuli
REDD1 specific RNA interference (RNAi) treated cells show a reduction of protein level on Western Blotting We reduced endogenous levels of REDD1 using a homemade shRNA construct designed to target REDD1 mRNA
Summary
Mechanistic target of rapamycin complex 1 (mTORC1) is a central signaling node in the cellular response to nutrient availability and growth factor signaling, initiating and regulating processes such as protein synthesis, ribosome biogenesis and de novo lipogenesis when active (reviewed by Laplante and Sabatini[1]). The switch is controlled by changes in mTORC1 activity that occur in response to variations in the availability of nutrients and energy requirements of the cell (see recent review by Albert and Hall[2]). The mechanism by which REDD1 acts to repress mTORC1 signaling has been under investigation for almost a decade[6]. These studies are focused on REDD1’s suppression of mTORC1 via its stimulation of the tuberous sclerosis complex 2 (TSC2); the method by which REDD1 activates TSC2 remained elusive[6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
