rabbit polyclonal anti keap1 antibody  (Proteintech)

Journal: eLife

Article Title: Lipid peroxidation and type I interferon coupling fuels pathogenic macrophage activation causing tuberculosis susceptibility

doi: 10.7554/eLife.106814

Figure Lengend Snippet: ( A ) Total levels of Nrf1, β-TrCP, and Keap1 proteins in B6 and B6.Sst1S BMDMs stimulated with TNF (10 ng/mL) for 8, 12, and 24 hr (western blotting). The experiment was repeated two times and shown the representative blot. ( B ) B6 and B6.Sst1S BMDMs stimulated with TNF (10 ng/mL) for 12 hr. Total NRF1 levels were evaluated using confocal microscopy. Scale bar = 20 μm. ( C ) B6 and B6.Sst1S BMDMs stimulated with TNF (10 ng/mL) for 12 hr. Nrf2 nuclear translocation was quantified using automated microscopy (Operetta CLS High Content Analysis System). Untreated samples were considered 100%. ( D ) The total antioxidant capacity of B6 and B6.Sst1S BMDMs was measured after TNF (10 ng/mL) stimulation. The percentage of induced antioxidant capacity upon TNF stimulation was plotted (Y axis). ( E and F ) B6 and B6.Sst1S BMDMs stimulated with TNF (10 ng/mL) for 8 and 12 hr. Hmox1 and Nqo1 expressions were quantified using qRT-PCR. ( G ) The heatmap of all genes related to response to oxidative stress (gene ontology category GO: 0006979). The heatmap was generated using FPKM values obtained using bulk RNAseq of B6.Sst1S and B6 BMDMs after 12 hr of TNF stimulation. For heatmap generation, FPKM values were scaled using Z-scores for each tested gene. The data are presented as means ± standard deviation (SD) from three to five samples per experiment, representative of three independent experiments. The statistical significance was performed by two-way ANOVA using Tukey’s multiple comparison test (Panels C-F). Significant differences are indicated with asterisks (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). Figure 2—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Antibody , Rabbit polyclonal anti-KEAP1 antibody (reactive to human, mouse, rat) , Proteintech Group Inc , Cat# 10503–2-AP, RRID: AB_2132625 , WB (1:5000).

Techniques: Western Blot, Confocal Microscopy, Translocation Assay, Microscopy, High Content Screening, Quantitative RT-PCR, Generated, Standard Deviation, Comparison

Journal: EMBO Reports

Article Title: KEAP1 retention in phase-separated p62 bodies drives liver damage under autophagy-deficient conditions

doi: 10.1038/s44319-025-00483-9

Figure Lengend Snippet: ( A ) Immunofluorescence microscopy. Primary cultured hepatocytes isolated from 5-week-old Atg7 flox/flox and Atg7 flox/flox ;Alb- Cre mice were immunostained with anti-p62 antibody. Scale bars: 10 μm. ( B ) Electron microscopy. Representative electron micrographs of cytoplasmic regions in primary cultured hepatocytes from 5-week-old Atg7 flox/flox ;Alb- Cre mice. Boxed region is displayed at higher magnification. Typical p62 bodies, along with concentric membranous structures associated with the endoplasmic reticulum (ER), accumulated in Atg7 -deficient hepatocytes. Arrow indicates the concentric membranous structure, while arrowheads indicate p62 bodies. Scale bars: 5 μm; 500 nm. ( C ) Immunoblot analysis. Wild-type ATG7 or the active-site mutant ATG7 C572S was introduced into Atg7 -knockout primary cultured hepatocytes (isolated from 5-week-old Atg7 flox/flox ;Alb- Cre mice) using an adenoviral system. Cell lysates were collected at the indicated time points after infection and subjected to immunoblot analysis with the specified antibodies. The bar graphs present the quantitative densitometric analysis of p62, Ser351-phosphorylated p62, and KEAP1, normalized to GAPDH ( n = 3). Data are presented as means ± s.e. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s test. ( D ) Immunofluorescence microscopy. Wild-type ATG7 or the active-site mutant ATG7 C572S was introduced into Atg7 -knockout primary cultured hepatocytes (isolated from 5-week-old Atg7 flox/flox ;Alb- Cre mice) using an adenoviral system. Immunostaining was performed at the indicated time points after infection with a p62 antibody. The size and number of p62 bodies per cell were quantified ( n = 500 cells). Horizontal bars indicate medians, boxes represent the interquartile range (25th–75th percentiles), and whiskers extend to 1.5× the interquartile range; individual outliers are shown as points. Statistical analysis was performed using Welch’s t test. Scale bars: 10 μm (main panels), 1 μm (inset panels). Scale bars: 10 μm. .

Article Snippet: Sections were blocked and incubated for 2 days at 4 °C with the following primary antibodies: mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan, 1:2000), rabbit polyclonal antibody against KEAP1 (10503-2-AP, Proteintech Group, 1:1000), and/or rabbit polyclonal antibody against CK19 (10712-1-AP, Proteintech Group, 1:1000) followed by Alexa594-Donkey anti-mouse IgG (711-545-152, Jackson ImmunoResearch) and/or Alexa488-Donkey anti-rabbit IgG (715-585-151, Jackson ImmunoResearch).

Techniques: Immunofluorescence, Microscopy, Cell Culture, Isolation, Electron Microscopy, Western Blot, Mutagenesis, Knock-Out, Infection, Immunostaining

Journal: EMBO Reports

Article Title: KEAP1 retention in phase-separated p62 bodies drives liver damage under autophagy-deficient conditions

doi: 10.1038/s44319-025-00483-9

Figure Lengend Snippet: ( A , B ) Immunohistofluorescence analysis. Liver sections from Atg7 flox/flox , Atg7 flox/flox ; p62 S351A/S351A , Atg7 flox/flox ;Alb- Cre , and Atg7 flox/flox ;Alb- Cre ; p62 S351A/S351A mice ( A ) and from Atg7 flox/flox , Atg7 flox/flox ; p62 T352A/T352A , Atg7 flox/flox ;Alb- Cre , and Atg7 flox/flox ;Alb- Cre ; p62 T352A/T352A mice ( B ) at 3 month-old were immunostained with anti-p62 (left panels) and anti-KEAP1 (middle panels) antibodies. The right panels show the merged images of p62 (red) and KEAP1 (green). Arrowheads indicate examples of p62 bodies. Bars: 10 μm. The graph shows the mean intensity of KEAP1 in p62 bodies in the livers of each genotype ( n = 4) mice. Vertical bars are means ± s.e. Statistical analysis was performed by Welch’s t test. ( C ) Schematic diagram of KEAP1 retention within p62 bodies in three mouse lines: Atg7 flox/flox ;Alb- Cre ( Atg7 -KO), Atg7 flox/flox ;Alb- Cre ; p62 S351A/S351A ( Atg7 -KO; p62 S351A ) and Atg7 flox/flox ;Alb- Cre ; p62 T352A/T352A ( Atg7 -KO; p62 T352A ) mice. .

Article Snippet: Sections were blocked and incubated for 2 days at 4 °C with the following primary antibodies: mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan, 1:2000), rabbit polyclonal antibody against KEAP1 (10503-2-AP, Proteintech Group, 1:1000), and/or rabbit polyclonal antibody against CK19 (10712-1-AP, Proteintech Group, 1:1000) followed by Alexa594-Donkey anti-mouse IgG (711-545-152, Jackson ImmunoResearch) and/or Alexa488-Donkey anti-rabbit IgG (715-585-151, Jackson ImmunoResearch).

Techniques: Immunohistofluorescence

Journal: EMBO Reports

Article Title: KEAP1 retention in phase-separated p62 bodies drives liver damage under autophagy-deficient conditions

doi: 10.1038/s44319-025-00483-9

Figure Lengend Snippet: ( A , B ) Immunoblot analysis. Livers from cont., p62 S351A , Atg7 -KO, and Atg7 -KO; p62 S351A mice ( A ) and from cont., p62 T352A , Atg7 -KO, and Atg7 -KO; p62 T352A mice ( B ) at 3 month-old were separated into nuclear and cytoplasmic fractions. The cytoplasmic fraction was subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies. Cont., in which ATG7 is efficiently expressed at similar level of the wild-type mice were used as control. Graph shows the results of quantitative densitometric analysis of ubiquitinated proteins, KEAP1, p62 and Ser351-phosphorylated p62 relative to the whole protein content estimated using Ponceau-S staining ( n = 3). Data are means ± s.e. Statistical analysis was performed by Tukey test after one-way ANOVA. Asterisks indicate non-specific bands, as they did not increase in the Atg7 knockout background. .

Article Snippet: Sections were blocked and incubated for 2 days at 4 °C with the following primary antibodies: mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan, 1:2000), rabbit polyclonal antibody against KEAP1 (10503-2-AP, Proteintech Group, 1:1000), and/or rabbit polyclonal antibody against CK19 (10712-1-AP, Proteintech Group, 1:1000) followed by Alexa594-Donkey anti-mouse IgG (711-545-152, Jackson ImmunoResearch) and/or Alexa488-Donkey anti-rabbit IgG (715-585-151, Jackson ImmunoResearch).

Techniques: Western Blot, SDS Page, Control, Staining, Knock-Out

Journal: EMBO Reports

Article Title: KEAP1 retention in phase-separated p62 bodies drives liver damage under autophagy-deficient conditions

doi: 10.1038/s44319-025-00483-9

Figure Lengend Snippet: ( A , B ) Immunoblot analysis. Wild-type p62 and indicated p62 mutants were introduced into p62 -knockout ( A ) or p62 and FIP200 -double knockout ( B ) Huh-1 cells, and the cell lysates were subjected to immunoblot analysis with indicated antibodies. Bar graph shows the results of quantitative densitometric analysis of KEAP1 relative to the whole protein content estimated using Ponceau-S staining ( n = 3). Data are means ± s.e. Statistical analysis was performed by Tukey test after one-way ANOVA. ( C ) Immunofluorescence microscopy. Huh-1 cells indicated in ( A ) were immunostained with the indicated antibodies. Scale bars, 10 μm (main panels). The graph shows the mean intensity of KEAP1 in p62 bodies comprising of wild-type p62 ( n = 2469), p62 S349E ( n = 1057), p62 S349A ( n = 2992), or p62 T350A ( n = 2033). Data are means ± s.e. Statistical analysis was performed by Tukey test after one-way ANOVA. ( D ) Immunofluorescence microscopy. Huh-1 cells indicated in ( B ) were immunostained with the indicated antibodies. Scale bars, 10 μm (main panels). The graph shows the mean intensity of KEAP1 in p62 bodies comprising of wild-type p62 ( n = 2892), p62 S349E ( n = 2611), p62 S349A ( n = 5191), or p62 T350A ( n = 3522). Data are means ± s.e. Statistical analysis was performed by Tukey test after one-way ANOVA. .

Article Snippet: Sections were blocked and incubated for 2 days at 4 °C with the following primary antibodies: mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan, 1:2000), rabbit polyclonal antibody against KEAP1 (10503-2-AP, Proteintech Group, 1:1000), and/or rabbit polyclonal antibody against CK19 (10712-1-AP, Proteintech Group, 1:1000) followed by Alexa594-Donkey anti-mouse IgG (711-545-152, Jackson ImmunoResearch) and/or Alexa488-Donkey anti-rabbit IgG (715-585-151, Jackson ImmunoResearch).

Techniques: Western Blot, Knock-Out, Double Knockout, Staining, Immunofluorescence, Microscopy

Journal: EMBO Reports

Article Title: KEAP1 retention in phase-separated p62 bodies drives liver damage under autophagy-deficient conditions

doi: 10.1038/s44319-025-00483-9

Figure Lengend Snippet: ( A , B ) Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) from cont. ( n = 10), p62 S351A ( n = 7), Atg7 -KO ( n = 13), and Atg7 -KO; p62 S351A ( n = 7) mice ( A ) and from cont. ( n = 3), p62 T352A ( n = 10), Atg7 -KO ( n = 5), and Atg7 -KO; p62 T352A ( n = 6) mice ( B ) were measured. IU/l, international units/liter. Data are means ± s.e. Statistical analysis was performed by Tukey test after one-way ANOVA. ( C ) Mechanism of liver injury development induced by autophagy suppression: KEAP1 retention via p62 body. .

Article Snippet: Sections were blocked and incubated for 2 days at 4 °C with the following primary antibodies: mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan, 1:2000), rabbit polyclonal antibody against KEAP1 (10503-2-AP, Proteintech Group, 1:1000), and/or rabbit polyclonal antibody against CK19 (10712-1-AP, Proteintech Group, 1:1000) followed by Alexa594-Donkey anti-mouse IgG (711-545-152, Jackson ImmunoResearch) and/or Alexa488-Donkey anti-rabbit IgG (715-585-151, Jackson ImmunoResearch).

Techniques: