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Cell Signaling Technology Inc antibodies against keap1
Antibodies Against Keap1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 435 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/antibodies+against+keap1/KEAP1+Rabbit+mAb/pm41703852-164-25-30
Average 96 stars, based on 435 article reviews

antibodies against keap1 - by Bioz Stars, 2026-10

96/100 stars

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Article Title: Irisin-mediated KEAP1 degradation alleviates oxidative stress and ameliorates pancreatitis.
Article Snippet: Oxidative stress (OS) injury is pivotal in acute pancreatitis (AP) pathogenesis, contributing to inflammatory cascades.. Irisin, a ubiquitous cytokine, exhibits antioxidant properties.. However, the role of irisin in AP remains inconclusive.

Article Title: The prostacyclin receptor PTGIR is a NRF2-dependent regulator of CD8 + T cell exhaustion
Article Snippet: Libraries were sequenced using a NovaSeq 6000 (Illumina) with 50 bp paired-end sequencing (5 × 10 7 reads per sample). .. Protein samples were prepared according to the methods section in ref. . Membranes were blocked for 1 h in 5% nonfat milk in 1× Tris-buffered saline with Tween 20 (TBST) at room temperature (RT) and incubated with primary antibodies against KEAP1 (Cell Signaling, cat. no. 8047, 1:500 v:v), NQO1 (Proteintech, cat. no. 11451-1-AP), HMOX1 (Proteintech, cat. no. 66743-1-ig) and LAMIN B1 (Cell Signaling, cat. no. 13435) overnight at 4 °C. .. Membranes were washed 3× for 5 min with 1× TBST and then incubated for 1 h at RT with the corresponding anti-rabbit immunoglobulin G (IgG; Cell Signaling, cat. no. 7074) or anti-mouse IgG (Cell Signaling, cat. no. 7076), horseradish peroxidase (HRP)-conjugated secondary antibody diluted in 5% nonfat milk.

Article Title: MiR-367-3p Alleviates Oxidative Stress Injury in the Ischemia/Reperfusion Injury Cell Model by Targeting Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4-mediated Keap1/Nrf2/ARE Pathway
Article Snippet: The lysates were precleared with control agarose resin from the Pierce Co‐Immunoprecipitation (Co‐IP) Kit (26,149, Thermoscientific). .. Subsequently, the supernatants were incubated with antibodies against Keap1 (1:1000, #8047, Cell signaling) and Nrf2 (1:1000, #12721, Cell signaling) or negative control immunoglobulin G (IgG) and protein IgG beads at 4°C overnight while being rotated. ..

Article Title: Nrf2 pathway potentially confers protection against cigarette smoke-induced sarcopenia in a mouse model
Article Snippet: Total protein was isolated from the skeletal muscle tissue using radioimmunoprecipitation assay (RIPA) lysis buffer (P0013C, Beyotime, China) and quantified. .. Subsequently, equal amount of protein sample was separated and transferred to polyvinylidene fluoride (PVDF) membrane, which was then incubated with primary antibodies against KEAP1 (#8047, 1/2000, Cell Signaling Technology, Danvers, MA, USA), NRF2 (#12721, 1/1000, Cell Signaling Technology, USA), autophagy-related gene 7 (ATG7, #8558, 1/1000, Cell Signaling Technology, USA), light chain 3 (LC3, ab192890, 1/2000, Abcam, Cambridge, UK) and housekeeping control Gapdh (ab181602, 1/5000, Abcam, UK) at 4 °C overnight. .. Additionally, the horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (ab205718, 1/5000, Abcam, UK) was applied for further incubation at ambient temperature for 1 h. Finally, the protein bands were visualized using an electrochemiluminescence (ECL) visualization kit (P0018S, Beyotime, China) and quantified by ImageJ (Version 5.0, Bio-Rad Laboratories, USA).

Article Title: The prostacyclin receptor PTGIR is a NRF2-dependent regulator of CD8 + T cell exhaustion.
Article Snippet: .. Membranes were blocked for 1 h in 5% nonfat milk in 1× Tris-buffered saline with Tween 20 (TBST) at room temperature (RT) and incubated with primary antibodies against KEAP1 (Cell Signaling, cat. no. 8047, 1:500 v:v), NQO1 (Proteintech, cat. no. 11451-1-AP), HMOX1 (Proteintech, cat. no. 66743-1-ig) and LAMIN B1 (Cell Signaling, cat. no. 13435) overnight at 4 °C. .. Membranes were washed 3× for 5 min with 1× TBST and then incubated for 1 h at RT with the corresponding anti-rabbit immunoglobulin G (IgG; Cell Signaling, cat. no. 7074) or anti-mouse IgG (Cell Signaling, cat. no. 7076), horseradish peroxidase (HRP)-conjugated secondary antibody diluted in 5% nonfat milk.

Article Title: Astragalus membranaceus sprouts and their unique constituents regulate reactive oxygen species production, inflammation-related senescence-associated secretory phenotype components, and extracellular matrix in fibroblasts.
Article Snippet: Aging involves a gradual decline in physiological functions and increased susceptibility to damage and disease.. Suppressing reactive oxygen species (ROS) production and inflammatory responses is critical for delaying agerelated cellular decline.. Astragalus membranaceus is one of the important health functional foods worldwide due to its health benefits, including antioxidant and immunomodulatory properties.

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Hepatocyte TM4SF5-mediated downregulation of protein KEAP1 via their binding is irrelevant to NRF2. Hepatocytes including TM4SF5-null cell lines [SNU449 lacking endogenously and Huh7 TM4SF5-KO via CRISPR/Cas9-mediated KO , TM4SF5-expressing cell lines (Hep3B and Huh7 Control expressing endogenously), or primary hepatocytes from C57BL/6 male mice (G) were cultured at subconfluent conditions. Cells were transfected either transiently or stably with the relevant cDNAs. Additionally, they were exposed to TSAHC (a TM4SF5-specific chalcone inhibitor) for the indicated durations at varying concentrations (I) or transfected with shRNA targeting either a control sequence (shNS) or shNRF2 (target sequences #1 or #2, see Table ). Whole cell extracts were prepared using lysis buffer containing Triton X-100, Brij58 (H), or Triton X-100 alone (A-G and I-L). Following normalization, proteins were processed for (immuno)precipitation using streptavidin-agarose beads, anti-NRF2 antibody, or anti-KEAP1 antibody, prior to immunoblot analysis for designated molecules. Data shown are representative of three independent experiments. See also and S2.

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Hepatocyte TM4SF5-mediated downregulation of protein KEAP1 via their binding is irrelevant to NRF2. Hepatocytes including TM4SF5-null cell lines [SNU449 lacking endogenously and Huh7 TM4SF5-KO via CRISPR/Cas9-mediated KO , TM4SF5-expressing cell lines (Hep3B and Huh7 Control expressing endogenously), or primary hepatocytes from C57BL/6 male mice (G) were cultured at subconfluent conditions. Cells were transfected either transiently or stably with the relevant cDNAs. Additionally, they were exposed to TSAHC (a TM4SF5-specific chalcone inhibitor) for the indicated durations at varying concentrations (I) or transfected with shRNA targeting either a control sequence (shNS) or shNRF2 (target sequences #1 or #2, see Table ). Whole cell extracts were prepared using lysis buffer containing Triton X-100, Brij58 (H), or Triton X-100 alone (A-G and I-L). Following normalization, proteins were processed for (immuno)precipitation using streptavidin-agarose beads, anti-NRF2 antibody, or anti-KEAP1 antibody, prior to immunoblot analysis for designated molecules. Data shown are representative of three independent experiments. See also and S2.

Article Snippet: Immunohistochemistry (IHC) was performed with antibodies against Keap1 (NBP2-03319, Novus Biologicals), Nrf2 (NBP1-32822, Novus Biologicals), F4/80 (#70076, Cell Signaling Technology), Cd11b (#PA5-79533, Invitrogen), Ccl2 (#MA5-17040, Invitrogen), and α-SMA (A2547, Sigma-Aldrich).

Techniques: Binding Assay, CRISPR, Expressing, Control, Cell Culture, Transfection, Stable Transfection, shRNA, Sequencing, Lysis, Immunoprecipitation, Western Blot

Correlations among TM4SF5, KEAP1, and CD36 levels following extracellular lipid treatment. (A-H) Subconfluent Huh7 (A, B, D, E, F, G, H, and I) and SNU449 (B, C) hepatocytes, either left unmodified or subjected to knockout (KO), were stably or transiently transfected with the specified cDNAs. For KEAP1 suppression, cells were treated with doxycycline (DOX) to induce silencing (G). In selected experiments, a lipid mixture (LM, 5%) was administered for 24 h. Following treatment, whole cell lysates (WCL) were prepared using lysis buffer containing Triton X-100. Lysates were quantified and subjected to immunoblotting (A, B, C, and G) or (immuno)precipitation (D, E, F, and H) with streptavidin-agarose beads, anti-Flag, or anti-KEAP1 antibodies, followed by immunoblotting for the indicated proteins. (I) Huh7 TM4SF5-KO cells complemented with mCherry-TM4SF5 WT were replated onto collagen I-precoated slide glasses and treated with either vehicle or lipid mixture prior to immunostaining and imaging. Scale bar: 100 μm. Data represent three independent experiments. See also .

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Correlations among TM4SF5, KEAP1, and CD36 levels following extracellular lipid treatment. (A-H) Subconfluent Huh7 (A, B, D, E, F, G, H, and I) and SNU449 (B, C) hepatocytes, either left unmodified or subjected to knockout (KO), were stably or transiently transfected with the specified cDNAs. For KEAP1 suppression, cells were treated with doxycycline (DOX) to induce silencing (G). In selected experiments, a lipid mixture (LM, 5%) was administered for 24 h. Following treatment, whole cell lysates (WCL) were prepared using lysis buffer containing Triton X-100. Lysates were quantified and subjected to immunoblotting (A, B, C, and G) or (immuno)precipitation (D, E, F, and H) with streptavidin-agarose beads, anti-Flag, or anti-KEAP1 antibodies, followed by immunoblotting for the indicated proteins. (I) Huh7 TM4SF5-KO cells complemented with mCherry-TM4SF5 WT were replated onto collagen I-precoated slide glasses and treated with either vehicle or lipid mixture prior to immunostaining and imaging. Scale bar: 100 μm. Data represent three independent experiments. See also .

Techniques: Knock-Out, Stable Transfection, Transfection, Lysis, Western Blot, Immunoprecipitation, Immunostaining, Imaging

TM4SF5-mediated stabilization of KEAP1 in lipid-treated hepatocytes promotes oxidative stress and hepatic inflammation. (A-F) Subconfluent Huh7 Control or Huh7 TM4SF5-KO hepatocyte variants, either stably or transiently transfected with the indicated cDNAs, were analyzed for ROS production using flow cytometry following DCFDA staining. Cells received vehicle treatment (-), PA alone (B, D, and F), lipid mixture (LM, C), or DOX to induce Keap1 knockdown (shKEAP1 #2 or #3 , see Table , C and D). In some experiments, Huh7 cell variants were transfected with siNS (non-specific sequences as a control) or siNRF2 (targeting sequence #1 or #2, see Table ) for 24 h. Cells were then treated with DOX for shKEAP1 #2 induction for 24 h, followed by PA for 4 h prior to ROS analysis by flow cytometry (D). Cells were also exposed to H 2 O 2 at the indicated concentrations and durations before assessment of ROS levels (E). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns = non-significant, unpaired Student's t test or two-way ANOVA. Data are expressed as mean ± SEM. (G) Subconfluent SNU449 hepatocytes stably expressing empty vector (SNU449 EV ) or TM4SF5 (SNU449 TM4SF5 ) were harvested to perform qRT-PCR for the indicated molecules. (H) Subconfluent Huh7 Control cells were treated in the absence (-) or presence (+) of DOX to induce KEAP1 knockdown (shKEAP1 #2 ) for 24 h and then exposed to PA (100 μM) for 4 h before being collected for immunoblot analysis of the indicated molecules. (I) Murine AML12 cells stably transfected with EV or TM4SF5 were treated with 5% LM for 24 h, harvested, and subjected to qRT-PCR for the indicated molecules. Data shown are representative of three independent experiments. See also .

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: TM4SF5-mediated stabilization of KEAP1 in lipid-treated hepatocytes promotes oxidative stress and hepatic inflammation. (A-F) Subconfluent Huh7 Control or Huh7 TM4SF5-KO hepatocyte variants, either stably or transiently transfected with the indicated cDNAs, were analyzed for ROS production using flow cytometry following DCFDA staining. Cells received vehicle treatment (-), PA alone (B, D, and F), lipid mixture (LM, C), or DOX to induce Keap1 knockdown (shKEAP1 #2 or #3 , see Table , C and D). In some experiments, Huh7 cell variants were transfected with siNS (non-specific sequences as a control) or siNRF2 (targeting sequence #1 or #2, see Table ) for 24 h. Cells were then treated with DOX for shKEAP1 #2 induction for 24 h, followed by PA for 4 h prior to ROS analysis by flow cytometry (D). Cells were also exposed to H 2 O 2 at the indicated concentrations and durations before assessment of ROS levels (E). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns = non-significant, unpaired Student's t test or two-way ANOVA. Data are expressed as mean ± SEM. (G) Subconfluent SNU449 hepatocytes stably expressing empty vector (SNU449 EV ) or TM4SF5 (SNU449 TM4SF5 ) were harvested to perform qRT-PCR for the indicated molecules. (H) Subconfluent Huh7 Control cells were treated in the absence (-) or presence (+) of DOX to induce KEAP1 knockdown (shKEAP1 #2 ) for 24 h and then exposed to PA (100 μM) for 4 h before being collected for immunoblot analysis of the indicated molecules. (I) Murine AML12 cells stably transfected with EV or TM4SF5 were treated with 5% LM for 24 h, harvested, and subjected to qRT-PCR for the indicated molecules. Data shown are representative of three independent experiments. See also .

Techniques: Control, Stable Transfection, Transfection, Flow Cytometry, Staining, Knockdown, Sequencing, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot

In vivo MASH-associated fibrosis model involves TM4SF5-dependent KEAP1 stabilization. (A-E) WT, Alb -TG Tm4sf5-Flag , and Tm4sf5 -/- KO C57BL/6 male mice (n=4~5, 8-week-old) were fed NCD or HFD without or with CCl 4 IP injection (HFDCCl 4 ) in the presence of vehicle DMSO or TSAHC treatment (IP injection, twice per week) for an additional 12 weeks until 20 weeks of age (A), after which they were euthanized for liver analysis using H&E, Masson's trichrome (M-T), Oil Red O (ORO), dihydroethidium (DHE) staining (B), immunohistochemistry (C), or immunoblotting (D and E). Scale bar: 100 μm. (F) Livers from animals fed only NCD or HFD for 12 weeks were processed to prepare whole tissue extracts for subsequent immunoblotting targeting the indicated molecules. (G) Liver tissues from animals treated as in (A) were analyzed by qRT-PCR for the target molecules. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, unpaired Student's t test or two-way ANOVA. Data are presented as mean ± SEM. Data shown are representative of three independent experiments. See also .

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: In vivo MASH-associated fibrosis model involves TM4SF5-dependent KEAP1 stabilization. (A-E) WT, Alb -TG Tm4sf5-Flag , and Tm4sf5 -/- KO C57BL/6 male mice (n=4~5, 8-week-old) were fed NCD or HFD without or with CCl 4 IP injection (HFDCCl 4 ) in the presence of vehicle DMSO or TSAHC treatment (IP injection, twice per week) for an additional 12 weeks until 20 weeks of age (A), after which they were euthanized for liver analysis using H&E, Masson's trichrome (M-T), Oil Red O (ORO), dihydroethidium (DHE) staining (B), immunohistochemistry (C), or immunoblotting (D and E). Scale bar: 100 μm. (F) Livers from animals fed only NCD or HFD for 12 weeks were processed to prepare whole tissue extracts for subsequent immunoblotting targeting the indicated molecules. (G) Liver tissues from animals treated as in (A) were analyzed by qRT-PCR for the target molecules. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, unpaired Student's t test or two-way ANOVA. Data are presented as mean ± SEM. Data shown are representative of three independent experiments. See also .

Techniques: In Vivo, Injection, Staining, Immunohistochemistry, Western Blot, Quantitative RT-PCR

Both murine and human MASH models demonstrate involvement of Tm4sf5, Keap1, and CD36, likely independent of the DNA-binding ability of NRF2. (A-E) WT, Nrf2 mut , Alb -TG Tm4sf5 ×Nrf2 mut , Tm4sf5 -/- KO×Nrf2 mut male C57BL/6 mice (n = 4~5, 8-week-old) were fed either NCD or HFD (60% kcal fat) alongside intraperitoneal CCl 4 injection (5 mg/kg, twice per week) for 12 weeks, after which tissues were examined using H&E staining or immunohistochemistry for Keap1 (B), or subjected to immunoblotting for selected proteins (C-E). (F and G) Analyses of GSE135251 dataset from either healthy controls or MASLD patients with varying NAS (from 0 to 4) or GSE48452 dataset was conducted to compare gene expression levels among normal, NAFL, and MASH patient samples stratified by NAS. The TM4SF5 High or TM4SF5 Low classification was determined based on the top 50% or bottom 50% expression levels of TM4SF5 mRNA in the cohort. (H) Human liver biopsy samples from control individuals and MASLD patients with different NAS (from 0 to 4) were used to obtain whole tissue extracts for immunoblotting of designated proteins. Because biopsy samples were very small (~ 7 mm circular pillar with 1 mm diameter), sufficient protein extracts for all targets were not always available, resulting in variable patient numbers analyzed in each immunoblot. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, one-way ANOVA. Data are reported as mean ± SEM. Findings presented represent three independent experimental replicates. See also .

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Both murine and human MASH models demonstrate involvement of Tm4sf5, Keap1, and CD36, likely independent of the DNA-binding ability of NRF2. (A-E) WT, Nrf2 mut , Alb -TG Tm4sf5 ×Nrf2 mut , Tm4sf5 -/- KO×Nrf2 mut male C57BL/6 mice (n = 4~5, 8-week-old) were fed either NCD or HFD (60% kcal fat) alongside intraperitoneal CCl 4 injection (5 mg/kg, twice per week) for 12 weeks, after which tissues were examined using H&E staining or immunohistochemistry for Keap1 (B), or subjected to immunoblotting for selected proteins (C-E). (F and G) Analyses of GSE135251 dataset from either healthy controls or MASLD patients with varying NAS (from 0 to 4) or GSE48452 dataset was conducted to compare gene expression levels among normal, NAFL, and MASH patient samples stratified by NAS. The TM4SF5 High or TM4SF5 Low classification was determined based on the top 50% or bottom 50% expression levels of TM4SF5 mRNA in the cohort. (H) Human liver biopsy samples from control individuals and MASLD patients with different NAS (from 0 to 4) were used to obtain whole tissue extracts for immunoblotting of designated proteins. Because biopsy samples were very small (~ 7 mm circular pillar with 1 mm diameter), sufficient protein extracts for all targets were not always available, resulting in variable patient numbers analyzed in each immunoblot. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, one-way ANOVA. Data are reported as mean ± SEM. Findings presented represent three independent experimental replicates. See also .

Techniques: Binding Assay, Injection, Staining, Immunohistochemistry, Western Blot, Gene Expression, Expressing, Control

Keap1 downregulation reverses TM4SF5-driven, MASH-associated fibrosis upon MCD diet. (A-D) WT, Alb -TG Tm4sf5-Flag , and Tm4sf5 -/- KO C57BL/6 male mice (n=4~5, 8-week-old) were maintained on an MCD diet for 3.5 weeks (A) and received intravenous injections of either PBS or siKeap1 (0.02 mg/kg, twice a week) prior to sacrifice for subsequent hepatic analyses, including body and liver weight measurements (B), and evaluation through H&E, Masson's trichrome (M-T), Oil Red O (ORO), or dihydroethidium (DHE) staining, immunohistochemistry (C), or immunoblotting (D) for specified proteins. Scale bar: 100 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, one-way ANOVA. All data are expressed as mean ± SEM.

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Keap1 downregulation reverses TM4SF5-driven, MASH-associated fibrosis upon MCD diet. (A-D) WT, Alb -TG Tm4sf5-Flag , and Tm4sf5 -/- KO C57BL/6 male mice (n=4~5, 8-week-old) were maintained on an MCD diet for 3.5 weeks (A) and received intravenous injections of either PBS or siKeap1 (0.02 mg/kg, twice a week) prior to sacrifice for subsequent hepatic analyses, including body and liver weight measurements (B), and evaluation through H&E, Masson's trichrome (M-T), Oil Red O (ORO), or dihydroethidium (DHE) staining, immunohistochemistry (C), or immunoblotting (D) for specified proteins. Scale bar: 100 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, one-way ANOVA. All data are expressed as mean ± SEM.

Techniques: Staining, Immunohistochemistry, Western Blot

Schematic model illustrating the modulation of KEAP1 by TM4SF5 under basal and pathological conditions. (Left) In healthy livers, TM4SF5 expression is very low and can be further upregulated by a hepatic inflammatory environment, presumably during early homeostatic conditions with minimal features of fatty liver. In this context, KEAP1 maintains regulation of NRF2 for antioxidant gene induction. In livers exhibiting balanced lipid catabolism and anabolism, or in the presence of extracellular lipids that do not induce MASH [e.g., HFD , low levels of tetraspanin TM4SF5 associate with KEAP1 and CD36, promoting KEAP1 ubiquitination and degradation and consequently increasing CD36 levels, thereby supporting homeostatic lipid metabolism. (Right) In situations of greater extracellular lipid overload, which can trigger MASH, TM4SF5 enhances the production of inflammatory cytokines and chemokines and increases ROS accumulation. Concurrently, TM4SF5 expression is elevated due to inflammation and further stabilizes KEAP1, facilitating ROS sensing and the induction of lipogenic genes. This leads to dysregulated lipid uptake, likely due to decreased CD36 levels, and an increase in NAS (from F1 to F4). Accordingly, dysregulated lipid metabolism and the pro-inflammatory environment, including ROS accumulation in TM4SF5-positive hepatocytes or livers, can drive MASH through TM4SF5-mediated KEAP1 stabilization, presumably independent of NRF2 levels or transcriptional function.

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Schematic model illustrating the modulation of KEAP1 by TM4SF5 under basal and pathological conditions. (Left) In healthy livers, TM4SF5 expression is very low and can be further upregulated by a hepatic inflammatory environment, presumably during early homeostatic conditions with minimal features of fatty liver. In this context, KEAP1 maintains regulation of NRF2 for antioxidant gene induction. In livers exhibiting balanced lipid catabolism and anabolism, or in the presence of extracellular lipids that do not induce MASH [e.g., HFD , low levels of tetraspanin TM4SF5 associate with KEAP1 and CD36, promoting KEAP1 ubiquitination and degradation and consequently increasing CD36 levels, thereby supporting homeostatic lipid metabolism. (Right) In situations of greater extracellular lipid overload, which can trigger MASH, TM4SF5 enhances the production of inflammatory cytokines and chemokines and increases ROS accumulation. Concurrently, TM4SF5 expression is elevated due to inflammation and further stabilizes KEAP1, facilitating ROS sensing and the induction of lipogenic genes. This leads to dysregulated lipid uptake, likely due to decreased CD36 levels, and an increase in NAS (from F1 to F4). Accordingly, dysregulated lipid metabolism and the pro-inflammatory environment, including ROS accumulation in TM4SF5-positive hepatocytes or livers, can drive MASH through TM4SF5-mediated KEAP1 stabilization, presumably independent of NRF2 levels or transcriptional function.

Techniques: Expressing, Ubiquitin Proteomics

Caffeic acid (CA) activate kelch-like ECH-associated protein 1/nuclear factor erythroid 2 related factor 2 (Keap1/Nrf2) signaling pathway. (A–E) Western blot and gray value analysis of Keap1 protein expression. (F) Western blot and gray value analysis of Nrf2 protein expression in the nucleus and cytoplasm. (G, H) Representative images of immunofluorescence staining of Keap1 (Green) and Nrf2 (Red). (I) Western blot and gray value analysis of heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate (NADPH) dehydrogenase quinone 1 (NQO1) protein expression. The results were normalized and are expressed as the mean ± standard deviation (SD) ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to the control group. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4′,6-diamidino-2′-phenylindole.

Journal: Journal of Pharmaceutical Analysis

Article Title: Caffeic acid alleviates myocardial ischemia-reperfusion injury by directly targeting Keap1 N532/M550 and promoting its degradation

doi: 10.1016/j.jpha.2025.101219

Figure Lengend Snippet: Caffeic acid (CA) activate kelch-like ECH-associated protein 1/nuclear factor erythroid 2 related factor 2 (Keap1/Nrf2) signaling pathway. (A–E) Western blot and gray value analysis of Keap1 protein expression. (F) Western blot and gray value analysis of Nrf2 protein expression in the nucleus and cytoplasm. (G, H) Representative images of immunofluorescence staining of Keap1 (Green) and Nrf2 (Red). (I) Western blot and gray value analysis of heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate (NADPH) dehydrogenase quinone 1 (NQO1) protein expression. The results were normalized and are expressed as the mean ± standard deviation (SD) ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to the control group. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4′,6-diamidino-2′-phenylindole.

Article Snippet: An antibody against Keap1 (10503-2-AP) was purchased from the proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Standard Deviation, Control

Caffeic acid (CA) induced kelch-like ECH-associated protein 1 (Keap1) degradation via p62-dependent autophagy. (A) Western blot and gray value analysis of Keap1 and p62 protein expression. (B) Immunofluorescence staining of p62 (Red) was analyzed by treating cells with different doses of CA. (C–E) Western blot and gray value analysis of Keap1 and LC3B-II protein expression were conducted both with and without the addition of MG132. (F–H) Western blot and gray value analysis of Keap1 and LC3B-II protein expression were conducted both with and without the addition of CQ (I) The LC3B-II (Red) expression level was detected by immunofluorescence analysis. The results were normalized and are expressed as the mean ± standard deviation (SD) ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to the control group. DAPI: 4′,6-diamidino-2′-phenylindole; CQ: chloroquin.

Journal: Journal of Pharmaceutical Analysis

Article Title: Caffeic acid alleviates myocardial ischemia-reperfusion injury by directly targeting Keap1 N532/M550 and promoting its degradation

doi: 10.1016/j.jpha.2025.101219

Figure Lengend Snippet: Caffeic acid (CA) induced kelch-like ECH-associated protein 1 (Keap1) degradation via p62-dependent autophagy. (A) Western blot and gray value analysis of Keap1 and p62 protein expression. (B) Immunofluorescence staining of p62 (Red) was analyzed by treating cells with different doses of CA. (C–E) Western blot and gray value analysis of Keap1 and LC3B-II protein expression were conducted both with and without the addition of MG132. (F–H) Western blot and gray value analysis of Keap1 and LC3B-II protein expression were conducted both with and without the addition of CQ (I) The LC3B-II (Red) expression level was detected by immunofluorescence analysis. The results were normalized and are expressed as the mean ± standard deviation (SD) ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to the control group. DAPI: 4′,6-diamidino-2′-phenylindole; CQ: chloroquin.

Article Snippet: An antibody against Keap1 (10503-2-AP) was purchased from the proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Standard Deviation, Control

Caffeic acid (CA) directly interacts with kelch-like ECH-associated protein 1 (Keap1) in vit r o . (A) Structure of CA and photo-affinity labeling probe (PAL-CA). (B) PAL-CA probe target fishing flowchart. (C) Silver staining of the PAL-CA complex in H9c2 cells. (D) Validation of Keap1 pulled down from mitochondria of the H9c2 cells with PAL-CA by Western blot. (E) Cellular thermal shift assay (CETSA) experiments of CA with Keap1 protein. (F) Size-exclusion chromatography analysis. The black and red lines represent the ultraviolet absorption of the standard and Keap1 proteins at 280 nM, respectively. (G) Surface plasmon resonance (SPR) experiments of CA with Keap1 protein. (H) Chemical structure of CA (top) and isothermal titration calorimetry (ITC) experiments (bottom). (I, J) SDS-PAGE gel and line graph were used to analyze the in vitro digestive stability of Keap1 under the action of trypsin.. (K) Native mass spectrometry analysis of apo Keap1. The Keap1 protein exists as monomers, dimers, and hexamers in solution (top). Enlarged view of the Keap1 protein dimer, including the P 1 and P 2 peaks (bottom). (L) Native mass spectrometry analysis of Keap1 with CA. After CA binds to the Keap1 protein, the monomers, dimers and hexamers exist in solution (top). Enlarged view of the increased dimerization that occurs after CA binds to the Keap1 protein (P 1 and P 2 peaks) (bottom). The results were normalized and are expressed as the mean ± standard deviation (SD) ( n = 3). DMSO: dimethyl sulfoxide.

Journal: Journal of Pharmaceutical Analysis

Article Title: Caffeic acid alleviates myocardial ischemia-reperfusion injury by directly targeting Keap1 N532/M550 and promoting its degradation

doi: 10.1016/j.jpha.2025.101219

Figure Lengend Snippet: Caffeic acid (CA) directly interacts with kelch-like ECH-associated protein 1 (Keap1) in vit r o . (A) Structure of CA and photo-affinity labeling probe (PAL-CA). (B) PAL-CA probe target fishing flowchart. (C) Silver staining of the PAL-CA complex in H9c2 cells. (D) Validation of Keap1 pulled down from mitochondria of the H9c2 cells with PAL-CA by Western blot. (E) Cellular thermal shift assay (CETSA) experiments of CA with Keap1 protein. (F) Size-exclusion chromatography analysis. The black and red lines represent the ultraviolet absorption of the standard and Keap1 proteins at 280 nM, respectively. (G) Surface plasmon resonance (SPR) experiments of CA with Keap1 protein. (H) Chemical structure of CA (top) and isothermal titration calorimetry (ITC) experiments (bottom). (I, J) SDS-PAGE gel and line graph were used to analyze the in vitro digestive stability of Keap1 under the action of trypsin.. (K) Native mass spectrometry analysis of apo Keap1. The Keap1 protein exists as monomers, dimers, and hexamers in solution (top). Enlarged view of the Keap1 protein dimer, including the P 1 and P 2 peaks (bottom). (L) Native mass spectrometry analysis of Keap1 with CA. After CA binds to the Keap1 protein, the monomers, dimers and hexamers exist in solution (top). Enlarged view of the increased dimerization that occurs after CA binds to the Keap1 protein (P 1 and P 2 peaks) (bottom). The results were normalized and are expressed as the mean ± standard deviation (SD) ( n = 3). DMSO: dimethyl sulfoxide.

Article Snippet: An antibody against Keap1 (10503-2-AP) was purchased from the proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Labeling, Silver Staining, Biomarker Discovery, Western Blot, Thermal Shift Assay, Size-exclusion Chromatography, SPR Assay, Isothermal Titration Calorimetry, SDS Page, In Vitro, Mass Spectrometry, Standard Deviation

The complexed crystal structure confirms the interaction sites of caffeic acid (CA) with kelch-like ECH-associated protein 1 (Keap1). (A) Schematic design of the experiments. (B) Gel filtration traces of Keap1 and Keap1 gel filtered with CA with a Superdex 200 10/300 Increase column and superimposed on the chromatogram of two standard protein markers (75 kDa and 44 kDa). (C) Kelch crystal diagram. (D) The overall structure of the complex of the Kelch domain with CA. Two orthogonal views are shown. (E) The CA molecule and surrounding residues responsible for its binding are shown in ball-and-stick representation. The M550 and N532 residues of the Kelch domain interact with CA. (F) Isothermal titration calorimetry (ITC) experiments of N532A with Keap1. (G) Comparison of the mouse Kelch domain (PDB ID: 1X2J ) and the Kelch domain bound to CA (PDB ID: 7YEN ). The Kelch apo and Kelch-CA complexes are colored wheat and purple, respectively. (H) Protein-ligand complex structure of molecular dynamics (MD) simulations for wild type 5 ns (a), wild type 100 ns (b), N532A mutant (c), and M550A mutant (d) of Keap1 kelch domain. The ligand CA is shown as yellow sticks. (I) Multiple sequence alignment of Keap1 from different species. The red background represents extremely conserved residues, and the red font represents relatively conserved residues. H. sapiens : Homo sapiens; M. musculus : Mus musculus ; C. toad : Caucasian toad ; D. rerio : Danio rerio .DP: ?.

Journal: Journal of Pharmaceutical Analysis

Article Title: Caffeic acid alleviates myocardial ischemia-reperfusion injury by directly targeting Keap1 N532/M550 and promoting its degradation

doi: 10.1016/j.jpha.2025.101219

Figure Lengend Snippet: The complexed crystal structure confirms the interaction sites of caffeic acid (CA) with kelch-like ECH-associated protein 1 (Keap1). (A) Schematic design of the experiments. (B) Gel filtration traces of Keap1 and Keap1 gel filtered with CA with a Superdex 200 10/300 Increase column and superimposed on the chromatogram of two standard protein markers (75 kDa and 44 kDa). (C) Kelch crystal diagram. (D) The overall structure of the complex of the Kelch domain with CA. Two orthogonal views are shown. (E) The CA molecule and surrounding residues responsible for its binding are shown in ball-and-stick representation. The M550 and N532 residues of the Kelch domain interact with CA. (F) Isothermal titration calorimetry (ITC) experiments of N532A with Keap1. (G) Comparison of the mouse Kelch domain (PDB ID: 1X2J ) and the Kelch domain bound to CA (PDB ID: 7YEN ). The Kelch apo and Kelch-CA complexes are colored wheat and purple, respectively. (H) Protein-ligand complex structure of molecular dynamics (MD) simulations for wild type 5 ns (a), wild type 100 ns (b), N532A mutant (c), and M550A mutant (d) of Keap1 kelch domain. The ligand CA is shown as yellow sticks. (I) Multiple sequence alignment of Keap1 from different species. The red background represents extremely conserved residues, and the red font represents relatively conserved residues. H. sapiens : Homo sapiens; M. musculus : Mus musculus ; C. toad : Caucasian toad ; D. rerio : Danio rerio .DP: ?.

Article Snippet: An antibody against Keap1 (10503-2-AP) was purchased from the proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Filtration, Binding Assay, Isothermal Titration Calorimetry, Comparison, Mutagenesis, Sequencing

Interaction analysis of caffeic acid (CA) analogs with kelch-like ECH-associated protein 1 (Keap1) Kelch . (A–D) The affinity of Keap1 Kelch binding with different components was detected by Biacore. (A) CGA with Keap1 Kelch , (B) CA-derivative with Keap1 Kelch , (C) Protocatechuic acid with Keap1 Kelch , D) Gallic acid with Keap1 Kelch . (E-H) The CA analogs and Keap1 Kelch interaction diagram. (E) Molecular docking of CGA with Keap1 Kelch , (F) Molecular docking of CA-derivative with Keap1 Kelch , (G) Molecular docking of protocatechuic acid with Keap1 Kelch , (H) Molecular docking of gallic acid with Keap1 Kelch , This figure is exported from the Ligplot software. Hydrogen bonds are shown as green dotted lines, while the spoked arcs represent residues making nonbonded contacts with the ligand. (I) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays of the toxic effects of chlorogenic acid (CGA) on H 2 O 2 -treated H9c2 cells. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to H 2 O 2 group. (J) Western blot and gray value analysis of Keap1 protein expression after treated with different concentrations of CGA. (K) Western blot and gray value analysis of Keap1 protein expression treated with different times of CGA. (L) Western blot and gray value analysis of Keap1 protein expression treated with different concentrations of 60 μM CGA. The results are expressed as the mean ± standard deviation (SD) ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to the control group.

Journal: Journal of Pharmaceutical Analysis

Article Title: Caffeic acid alleviates myocardial ischemia-reperfusion injury by directly targeting Keap1 N532/M550 and promoting its degradation

doi: 10.1016/j.jpha.2025.101219

Figure Lengend Snippet: Interaction analysis of caffeic acid (CA) analogs with kelch-like ECH-associated protein 1 (Keap1) Kelch . (A–D) The affinity of Keap1 Kelch binding with different components was detected by Biacore. (A) CGA with Keap1 Kelch , (B) CA-derivative with Keap1 Kelch , (C) Protocatechuic acid with Keap1 Kelch , D) Gallic acid with Keap1 Kelch . (E-H) The CA analogs and Keap1 Kelch interaction diagram. (E) Molecular docking of CGA with Keap1 Kelch , (F) Molecular docking of CA-derivative with Keap1 Kelch , (G) Molecular docking of protocatechuic acid with Keap1 Kelch , (H) Molecular docking of gallic acid with Keap1 Kelch , This figure is exported from the Ligplot software. Hydrogen bonds are shown as green dotted lines, while the spoked arcs represent residues making nonbonded contacts with the ligand. (I) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays of the toxic effects of chlorogenic acid (CGA) on H 2 O 2 -treated H9c2 cells. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to H 2 O 2 group. (J) Western blot and gray value analysis of Keap1 protein expression after treated with different concentrations of CGA. (K) Western blot and gray value analysis of Keap1 protein expression treated with different times of CGA. (L) Western blot and gray value analysis of Keap1 protein expression treated with different concentrations of 60 μM CGA. The results are expressed as the mean ± standard deviation (SD) ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to the control group.

Article Snippet: An antibody against Keap1 (10503-2-AP) was purchased from the proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Binding Assay, Software, Western Blot, Expressing, Standard Deviation, Control

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