plasmid pcdna3 1 flag nrf2  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: PtdIns(4,5)P 2 interacts with NRF2 in the nucleus. A , endogenous NRF2 was IPed from MDA-MB-231 cells, and PtdIns(4,5)P 2 was detected by fluorescent IB. IgG was used as a negative control. B , MDA-MB-231 cells were cultured from low confluency in media containing [ 3 H] myo -inositol or unlabeled myo -inositol. After 72 h, cells were treated with 200 μM DEM or vehicle for 4 h before being processed for IP against NRF2. NRF2 and PtdIns(4,5)P 2 were confirmed and quantified ( A ) by fluorescent WB and ImageJ before samples were resolved by SDS-PAGE and the gel sectioned corresponding to the NRF2 immunoblot. Gel sections from the indicated lanes were then dissolved and analyzed by liquid scintillation counting (LSC). Graph is mean ± SD C , HEK293FT cells were transiently transfected with FLAG-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. D and E , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( D ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( E ) is shown as mean ± SD of three independent experiments with n = 10 cells scored in each independent experiment. F and G , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( F ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( G ) is shown as mean ± SD of three independent experiments with n = 15 cells scored in each independent experiment. For all panels , n = 3 independent experiments; scale bar represents 5 μm. DAPI, 4',6-diamidino-2-phenylindole; DEM, diethyl maleate; HEK293FT, human embryonic kidney 293FT cell line; IB, immunoblotting; IF, immunofluorescence; IgG, immunoglobulin G; IP, immunoprecipitation; IPed, immunoprecipitated; NRF2, nuclear factor erythroid 2–related factor 2; PLA, proximity ligation assay; PtdIns(4,5)P 2 , phosphatidylinositol 4,5-bisphosphate; WB, Western blot.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Negative Control, Cell Culture, SDS Page, Western Blot, Transfection, Staining, Microscopy, Immunofluorescence, Immunoprecipitation, Proximity Ligation Assay

Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: PIPKIγ controls NRF2 stability. A and B , the expression of NRF2 was analyzed by IB in MDA-MB-231 cells with deletion of PIPKIα, PIPKIγ, PIPKIIα, or PIPKIIβ ( A ). The intensity of the immunoblots was quantified, and the graph is shown ( B ) as mean ± SD. C , KD of PIPKIγ using two shRNAs in HCT116 cells. Cells were treated with vehicle or 100 μM tBHQ for 4 h. The expression of NRF2 was analyzed by IB, and the graph is shown as mean ± SD. D , MDA-MB-231 cells (parental or with PIPKIγ deletion) were treated with 100 μM tBHQ for 4 h. NRF2 protein expression was analyzed by IB. E , MDA-MB-231 cells transfected with control siRNAs or siRNAs targeting PIPKIγ were treated with or without 10 μM MG132 for the indicated time. NRF2 protein levels were determined by IB and quantified ( B ) by ImageJ. For all panels , n = 3 independent experiments. KD, knockdown; IB, immunoblotting; NRF2, nuclear factor erythroid 2–related factor 2; PIPKIγ, phosphatidylinositol phosphate kinase γ; tBHQ, tertiary butylhydroquinone.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Expressing, Western Blot, Transfection, Control, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: PIPKIγ binds nuclear NRF2 . A and B , MDA-MB-231 ( A ) or HCT116 ( B ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB and quantified by ImageJ. C and D , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( C ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( D ) is shown as mean ± SD of three independent experiments with n = 10 cells scored in each independent experiment. E and F , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( E ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( F ) is shown as mean ± SD of three independent experiments with n = 15 cells scored in each independent experiment. G and H , MDA-MB-231 cells with or without PIPKIγ knockdown (KD) were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( G ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LASX (Leica). The experiments were repeated three times. The graph is shown as mean ± SD of n = 15 cells from one representative experiment ( H ). For all panels , n = 3 independent experiments; scale bar represents 5 μm. DAPI, 4',6-diamidino-2-phenylindole; DEM, diethyl maleate; IB, immunoblotting; IF, immunofluorescence; IPed, immunoprecipitated; NRF2, nuclear factor erythroid 2-related factor 2; PIPKIγ, phosphatidylinositol phosphate kinase γ; PLA, proximity ligation assay.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Staining, Microscopy, Knockdown, Western Blot, Immunofluorescence, Immunoprecipitation, Proximity Ligation Assay

Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: HSP27 interacts with NRF2 . A and B , endogenous NRF2 was IPed from MDA-MB-231 ( A ) or MCF7 ( B ) cells, and HSP27, αB-crystallin, HSP40, HSP60, and HSP90 were analyzed by IB. IgG was used as a negative control. C , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous NRF2 was IPed, and HSP27 was analyzed by IB. D , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous HSP27 was IPed, and NRF2 was analyzed by IB. E , quantification using ImageJ of co-IPed protein levels ( C and D ). F and G , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF staining against HSP27 and NRF2 ( F ). DAPI was used to stain nuclei. The images ( F ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The experiments were repeated three times, and the graph ( G ) is shown as mean ± SD of three independent experiments with n = 10 cells scored in each independent experiment. H and I , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between HSP27 and NRF2. DAPI was used to stain nuclei. The images ( H ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( I ) is shown as mean ± SD of three independent experiments with n = 15 cells scored in each independent experiment. For all panels , n = 3 independent experiments; scale bar represents 5 μm. DAPI, 4',6-diamidino-2-phenylindole; DEM, diethyl maleate; HSP27, heat shock protein 27; IB, immunoblotting; IF, immunofluorescence; IgG, immunoglobulin G; IPed, immunoprecipitated; NRF2, nuclear factor erythroid 2-related factor 2; PLA, proximity ligation assay.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Negative Control, Staining, Microscopy, Western Blot, Immunofluorescence, Immunoprecipitation, Proximity Ligation Assay

Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: PtdIns(4,5)P 2 binding to NRF2 promotes its interaction with sHSPs. A , schematic representation of NRF2 truncation mutants used in the study. B , recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB and the bound NRF2 mutants were quantified for HSP27 ( C ) and αB-crystallin ( D ) using ImageJ. E , recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. F and G , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 ( F ) or αB-crystallin ( G ) was analyzed by IB. For all panels , n = 3 independent experiments. HSP27, heat shock protein 27; PtdIns(4,5)P 2 , phosphatidylinositol 4,5-bisphosphate; sHSP, small heat shock protein.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Binding Assay, Recombinant, Mutagenesis, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: HSP27 stabilizes NRF2. A , MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27, and NRF2 expression was analyzed by IB 72 h later. The graph is shown as mean ± SD. B , RT–PCR analysis of NFE2L2 mRNA in MDA-MB-231 cells. NFE2L2 mRNA levels were normalized to GAPDH mRNA. The graph is shown as mean ± SD of n = 4 independent experiments. C , MDA-MB-231 cells transfected with control siRNAs or siRNAs targeting HSP27 were treated with or without 10 μM MG132 for the indicated time. NRF2 protein levels were determined by IB. D and E , HCT116 ( D ) or HS578 ( E ) cells were transiently transfected with control siRNAs or siRNAs targeting HSP27. About 68 h later, transfected cells were treated with vehicle or 100 μM tBHQ for an additional 4 h, and NRF2 expression was then analyzed by IB. The graph is shown as mean ± SD. F , A549 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27, and NRF2 expression was analyzed by IB 72 h later. The graph is shown as mean ± SD. G , 0.5 μg of recombinant His-tagged NRF2 and 9 μg KEAP1 were incubated with the indicated concentrations of HSP27. NRF2 was pulled down, and the bound KEAP1 and HSP27 were analyzed by IB. For all panels , n = 3 independent experiments unless otherwise indicated. HSP27, heat shock protein 27; IB, immunoblotting; KEAP1, Kelch-like ECH-associated protein 1; NRF2, nuclear factor erythroid 2–related factor 2; tBHQ, tertiary butylhydroquinone.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Transfection, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Recombinant, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: PIPKI γ and HSP27 regulate HO-1 expression, ROS levels, and oxidative stress–induced cell death. A , HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 was analyzed by IB and quantified by ImageJ ( C ). B , MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. C and D , ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( C ) or PIPKIγ ( D ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± SD. E and F , cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( E ) or PIPKIγ ( F ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± SD. G , a schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 , and sHSPs. For all panels , n = 3 independent experiments. DEM, diethyl maleate; HO-1, heme oxygenase-1; HSP27, heat shock protein 27; IB, immunoblotting; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NRF2, nuclear factor erythroid 2–related factor 2; PIPKIγ, phosphatidylinositol phosphate kinase γ; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; ROS, reactive oxygen species; sHSP, small heat shock protein.

Article Snippet: NRF2 plasmid (##36971) from Addgene was transiently transfected into HEK293FT cells using Lipofectamine 2000 (#11668019) from ThermoFisher.

Techniques: Expressing, Control, shRNA, Transfection, MTT Assay, Western Blot

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Left, Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or Myc-SDCBP-expressing vector in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( B ) Free heme level in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector ( n = 3). ( C ) Free heme level in scramble and in SDCBP - KO MDA-MB-231 cells ( n = 3). ( D ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or NRF2 (encoded by NFE2L2 )-expressing vector. HO-1 protein expression was considered as the positive control. ( E ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with a control or a HO-1 (encoded by HMOX1 )-expressing vector. ( F ) Western blot showing BACH1 protein expression in Hs578T cells transfected with scramble or HO-1 siRNA. ( G ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with the HO-1 or the catalytic inactive HO-1 mutant (H25A) plasmid. ( H ) Western blot showing BACH1 and SDCBP protein expression in MDA-MB-231 cells transfected with scramble or HOIL1 siRNA. ( I ) Western blot showing endogenous FBXO22 protein expression in several breast cancer cells. ( J ) Immunoprecipitation showing the interaction of BACH1 with FBXO22 in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ns: none specific. ( K ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in HEK293 cells transfected with the indicated plasmids. ( L ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in MDA-MB-231 cells transfected with the indicated plasmids. ( M ) In vivo ubiquitylation assay showing the decrease in FBXO22-mediated polyubiquitylation of BACH1 by SDCBP overexpression in HEK293 cells transfected with the indicated plasmids. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( A – C ). All experiments were repeated at least three times unless otherwise indicated. P values less than 0.05 were considered statistically significant.

Article Snippet: pcDNA3.1 FLAG NRF2 , Addgene , Cat#36971.

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, Positive Control, Mutagenesis, Immunoprecipitation, In Vivo, Ubiquitin Assay, Over Expression, Two Tailed Test