Journal: The Journal of Biological Chemistry
Article Title: Nrf2-and p53-inducible REDD2/DDiT4L/Rtp801L confers pancreatic β-cell dysfunction, leading to glucose intolerance in high-fat diet-fed mice
doi: 10.1016/j.jbc.2025.110271
Figure Lengend Snippet: Interaction of nuclear factor erythroid 2-related factor 2 (Nrf2) and p53 on the promoter of the regulated development and DNA damage response 2 ( Redd2 ) in INS-1 β-cells . A , INS-1 cells were transfected with Nrf2 and/or p53 expression vector by electroporation for 24 h and Redd2 expressions were determined. B–G , INS-1 cells were transfected with Nrf2 and/or p53 expression vector, a firefly luciferase reporter vector of wild type or mutant mouse Redd2 promoter (−2328/−1) (WT ( B ), EpRE2 mutant ( C ), p53RE1 mutant ( D ), or EpRE2/p53RE1 mutant ( E )), EpRE reporter vector (F), or 2xp53RE reporter (G) and the Renilla luciferase reporter vector (pGL4.74[ hRluc /TK] or pGL4.73[ hRluc /SV40]) for 24 h. Luciferase activities were determined using Dual-Luciferase reporter assay system. H – K , ChIP assay was performed using control IgG, anti-p-Nrf2 IgG, and anti-p-p53 IgG. The co-precipitated DNA was subjected to PCR using primers that amplify Redd2 promoter containing the EpRE2 ( H ) or p53RE1 ( J ) elements in rat INS-1 cells. Densitometry of relative amplified DNA bands containing the EpRE2 ( I ) and p53RE1 ( K ). L , GST pull-down assay with purified GST or GST-Myc3-Nrf2 and His-Flag-p53. Pulled down samples were analyzed with western blotting with anti-GST and anti-Flag antibodies. Data are expressed as mean ± SD (n = 4). Asterisk indicates statistically significant differences ( p < 0.05, Dunnett’s test). Different letters indicate statistically significant differences ( p < 0.05, Tukey-Kramer's test).
Article Snippet: Western blotting was performed using rabbit monoclonal anti-p70S6 kinase (#2708, Cell Signaling Technology, RRID: AB_390722 ), rabbit monoclonal anti-phospho-p70S6K (#9234, Cell Signaling Technology, RRID: AB_2269803 ), rabbit polyclonal anti-Nrf2 (sc-13032, Santa Cruz Biotechnology, RRID: AB_2263168 ), rabbit polyclonal anti-p53 (bs-0033R, Bioss, Boston, MA, USA, RRID: AB_10855396 ), rabbit polyclonal anti-phospho-p53 (# 9284, Cell Signaling Technology, RRID: AB_331464 ), mouse monoclonal anti-DDIT4L (DDIT-03, Thermo Fisher Scientific, RRID: AB_2745588 ), rabbit polyclonal anti-triosephosphate isomerase (TPI) , rabbit polyclonal anti-Histone H2B (07-371, Millipore, RRID: AB_310561 ), or mouse monoclonal anti-β-actin (66009-1-Ig, Proteintech, RRID: AB_2687938 ) as the primary antibodies, and HRP-conjugated goat anti-rabbit or goat anti-mouse antibodies (170-6515, Bio-Rad, Hercules, CA, USA, RRID: AB_11125142 or 170-6516, Bio-Rad, RRID: AB_11125547 ) as secondary antibodies.
Techniques: Transfection, Expressing, Plasmid Preparation, Electroporation, Luciferase, Mutagenesis, Reporter Assay, Control, Amplification, Pull Down Assay, Purification, Western Blot
Bioss is a verified supplier 
Bioss manufactures this product 
![Regulation of the regulated development and DNA damage response 2 ( Redd2 ) expression by nuclear factor erythroid 2-related factor 2 (Nrf2) and p53 in INS-1 β-cells. A and B , INS-1 cells transfected with Nrf2 ( A ) or p53 ( B ) expression vector were incubated for 12 h. Redd2 mRNA expression was determined with quantitative RT-PCR. C–E , INS-1 cells transfected with siNrf2 or sip53 were incubated with or without of 1 mM streptozotocin (STZ) for 6 h. The mRNA expressions of Nrf2 ( C ), p53 ( D ), and Redd2 ( E ) were determined using quantitative RT-PCR. F , Promoter analysis of putative electrophile response elements (EpREs) and putative p53 response elements (p53REs) in mouse Redd2 promoter (−2328/−1) region. G and H , INS-1 cells transfected with Nrf2 or p53 expression vector, a firefly luciferase reporter vector containing the wild-type (WT) or mutant Redd2 promoter (−2328/−1), and the Renilla luciferase reporter vector pGL4.73[ hRluc /SV40] for 24 h. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System. Data are expressed as mean ± SD (n = 4). Asterisks indicate statistically significant differences ( p < 0.05, Student's t test). Different letters indicate statistically significant differences ( p < 0.05, Tukey-Kramer's test).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6130/pmc12206130/pmc12206130__gr3.jpg)
![Interaction of nuclear factor erythroid 2-related factor 2 (Nrf2) and p53 on the promoter of the regulated development and DNA damage response 2 ( Redd2 ) in INS-1 β-cells . A , INS-1 cells were transfected with Nrf2 and/or p53 expression vector by electroporation for 24 h and Redd2 expressions were determined. B–G , INS-1 cells were transfected with Nrf2 and/or p53 expression vector, a firefly luciferase reporter vector of wild type or mutant mouse Redd2 promoter (−2328/−1) (WT ( B ), EpRE2 mutant ( C ), p53RE1 mutant ( D ), or EpRE2/p53RE1 mutant ( E )), EpRE reporter vector (F), or 2xp53RE reporter (G) and the Renilla luciferase reporter vector (pGL4.74[ hRluc /TK] or pGL4.73[ hRluc /SV40]) for 24 h. Luciferase activities were determined using Dual-Luciferase reporter assay system. H – K , ChIP assay was performed using control IgG, anti-p-Nrf2 IgG, and anti-p-p53 IgG. The co-precipitated DNA was subjected to PCR using primers that amplify Redd2 promoter containing the EpRE2 ( H ) or p53RE1 ( J ) elements in rat INS-1 cells. Densitometry of relative amplified DNA bands containing the EpRE2 ( I ) and p53RE1 ( K ). L , GST pull-down assay with purified GST or GST-Myc3-Nrf2 and His-Flag-p53. Pulled down samples were analyzed with western blotting with anti-GST and anti-Flag antibodies. Data are expressed as mean ± SD (n = 4). Asterisk indicates statistically significant differences ( p < 0.05, Dunnett’s test). Different letters indicate statistically significant differences ( p < 0.05, Tukey-Kramer's test).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6130/pmc12206130/pmc12206130__gr4.jpg)