anti keap1 mouse  (Santa Cruz Biotechnology)

Journal: Scientific Reports

Article Title: Curcumin preserves bone health compromised by diabetes by inhibiting osteoporosis through regulation of the SIRT3/FoxO3a signalling pathway

doi: 10.1038/s41598-025-15165-8

Figure Lengend Snippet: Curcumin upregulates the expression of the oxidative stress-related proteins Nrf2 and SIRT3 in bone tissue cells. Histograms indicate the SIRT3 protein expression levels relative to the control group. Values are expressed as mean ± standard deviation for three independent experiments ( n = 3). * p < 0.05, *** p < 0.001 vs. control group, # p < 0.05, ## p < 0.01 vs. HG group.

Article Snippet: Curcumin (Solarbio), mouse anti-SIRT3 antibody (Proteintech) , mouse anti-FoxO3a antibody (Proteintech), mouse anti-cytochrome c antibody (Proteintech), mouse anti-NRF2 antibody (Proteintech), mouse anti-NOX2 antibody (Proteintech), mouse anti-caspase 3 antibody (Proteintech), mouse anti-SOD2 antibody (Proteintech), rabbit anti-mouse SIRT3 antibody (Proteintech) Proteintech), rabbit anti-mouse Bcl2 antibody (Proteintech), mouse anti-β-actin antibody (Beijing Zhongyi Jinqiao Company), sodium pentobarbital (Sigma), paraformaldehyde (Xi’an Reagent Factory), α-D-glucose (Sigma), anti-osteocalcin (OCN) antibody (Merck & Millipore ), NQO1 (Proteintech), mouse osteoprotegerin (OPG) antibody (R&D), minimum essential medium alpha (α-MEM) (Bioind), dimethyl sulfoxide Hybri-Max (Sigma), Flow Cytometry Kit (Jiangsu Biyuntian), osteogenic induction complete medium (Pythonbio), Mitochondrial Membrane Potential Assay Kit (Jiangsu Biyuntian), ROS Reactive Oxygen Species Assay Kit (Jiangsu Biyuntian).

Techniques: Expressing, Control, Standard Deviation

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: ( A ) Location of differential accessible ATAC-seq peaks in NC and Hmgb2 -cKO CD8 + T cells. 3′UTR, 3′ untranslated region; 5′UTR, 5′ untranslated region. ( B ) Chromatin accessibility changes of genes associated with effector function and mitochondrial transcription factors. ( C ) The correlation between Hmgb2 , Tfam , and Tfb1m expression in CD8 + T cells from murine scRNA-seq data. ( D ) mRNA levels of Keap1 and Nfe2l2 in NC and Hmgb2 -cKO CD8 + T cells ( n = 6). ( E ) mRNA levels of ARE genes in NC and Hmgb2 -cKO CD8 + T cells ( n = 3). ( F ) Protein changes of KEAP1 and NRF2 after Hmgb2 knockout. ( G ) The ubiquitination of NRF2 after Hmgb2 knockout. IB, immunoblot. ( H ) Protein changes of KEAP1 and NRF2 after stimulation of mouse recombinant HMGB2 protein and IN-1. NC CD8 + T cells were treated with recombinant HMGB2 protein (500 ng/ml) and/or IN-1 (10 μM) for 48 hours. ( I ) The ubiquitination of NRF2 after cell treatment as in (H). ( J ) Immunofluorescence staining of spontaneous HCC tissues in NC and Hmgb2 -cKO mice. NRF2 + KEAP1 − CD8 + T cells were labeled. Scale bars, 20 μm (left) and 10 μm (right). HP, high power field. ( K ) Seahorse extracellular flux analysis of OCR in different CD8 + T cell groups ( n = 6 to 8) after cell treatment as in (H). ( L ) Quantification of seahorse extracellular flux analysis of OCR of different CD8 + T cells as in (K). Data are presented as the means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Pearson test for (C) Student’s t test for (D), (E), and (J). Two-way ANOVA test for (L).

Article Snippet: Anti-human/mouse KEAP1 , 10503-2-AP , Proteintech, China.

Techniques: Expressing, Knock-Out, Ubiquitin Proteomics, Western Blot, Recombinant, Immunofluorescence, Staining, Labeling

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: Markers and article numbers of antibodies. PE, phycoerythrin; FITC, fluorescein isothiocyanate; APC, antigen-presenting cell; HRP, horseradish peroxidase; mAb, monoclonal antibody.

Article Snippet: Anti-human/mouse KEAP1 , 10503-2-AP , Proteintech, China.

Techniques: Ubiquitin Proteomics, Purification, In Vivo

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: Names and sequence of primers. F, forward; R, reverse.

Article Snippet: Anti-human/mouse KEAP1 , 10503-2-AP , Proteintech, China.

Techniques: Sequencing

Journal: bioRxiv

Article Title: A pharmacological modality to sequester homomeric proteins

doi: 10.1101/2025.03.05.641597

Figure Lengend Snippet: a. Chemical structures of Keap1-targeting PINCHs. EC 50 values for the active compounds are shown in parentheses. b. WB of EL229’s effect on Keap1 in OCI-AML2 cells’ soluble and insoluble phases; 20 h treatment. Full gels are shown in Fig. S10. c. WB analysis of additional active Keap1-targeting PINCHs in OCI-AML2 cells; 20 h. d. EL133 affects Keap1 in the soluble and insoluble OCI-AML2 cell phases, in a time- and dose-dependent manner. e. WB band quantification and calculated EC 50 values from n=3 independent experiments. f. Immunostaining of U2OS cells following treatment with 250nM Bardoxolone (left) or 250nM EL133 (right). Keap1 is colored in Magenta; DAPI staining in Cyan. g. Quantification of Keap1 ‘puncta’ in the two treatments shows a significant difference in volume ( p <0.0001, two-tailed Mann-Whitney test, n=129 cells analysed in 2 independent experiments, additional images are shown in Fig. S7). Error-bars show 95% confidence interval. h. Immunostaining of U2OS cells following a 20 hour treatment with 2.5 μM BODIPY-conjugated PINCH EL256, and its structure. Keap1 is colored in Magenta; DAPI staining in Cyan. i. Global proteomics analysis of OCI-AML2 cells soluble lysate, showing significant downregulation of Keap1 following treatment with EL133 (2.5 μM; 20 h), compared to DMSO. The dotted lines mark a 1.75-fold reduction in protein level (vertical) and p =0.05 significance (horizontal). j. rt-PCR analysis of NQO1 expression after a 20h treatment of OCI-AML2 cells with DMSO, EL133 or BDX at 250 nM, followed by washing of the cells (representative results following two independent experimental replicates with two technical replicates each). Error-bars show the standard deviation. k. Lattice light-sheet microscope live imaging of U2OS cells, immediately following treatment with 2.5 μM EL256. Time post-treatment is indicated in hr:min.

Article Snippet: Incubation with primary antibodies (mouse anti-BCL-6 (sc-7388, Santa Cruz, 1:500, overnight at 4 °C), mouse anti-Keap1 (sc-365626, Santa Cruz, 1:500, overnight at 4 °C), rabbit anti-c-Myc (#5605, Cell Signaling, 1:1000, overnight at 4°C), rabbit anti-p62 (ab155686, Abcam, 1:1000, overnight at 4 °C) and mouse anti-beta-actin (#3700, Cell Signaling, 1:1000, 1 h at room temperature) was performed, then the membranes were washed and incubated with an HRP-conjugated secondary antibody (mouse #7076/rabbit #7074, Cell Signaling) at room temperature for 1 hour.

Techniques: Immunostaining, Staining, Two Tailed Test, MANN-WHITNEY, Reverse Transcription Polymerase Chain Reaction, Expressing, Standard Deviation, Microscopy, Imaging

Journal: bioRxiv

Article Title: A pharmacological modality to sequester homomeric proteins

doi: 10.1101/2025.03.05.641597

Figure Lengend Snippet: a. WB analyses of Keap1-targeting PINCHs in OCI-AML2 cells at 20 h treatments. All full gels are available in Fig. S10,11. b. Global proteomics analysis of OCI-AML2 cells treated with 2.5μM EL133/DMSO, lysed by sonication prior to sample analysis. c. OCI-AML2 cells pre-treated with Bortezomib or MLN4924 for 1 hour, then with EL133 or NC-1, a BTK-targeting PROTAC , for an additional 7 hours.

Article Snippet: Incubation with primary antibodies (mouse anti-BCL-6 (sc-7388, Santa Cruz, 1:500, overnight at 4 °C), mouse anti-Keap1 (sc-365626, Santa Cruz, 1:500, overnight at 4 °C), rabbit anti-c-Myc (#5605, Cell Signaling, 1:1000, overnight at 4°C), rabbit anti-p62 (ab155686, Abcam, 1:1000, overnight at 4 °C) and mouse anti-beta-actin (#3700, Cell Signaling, 1:1000, 1 h at room temperature) was performed, then the membranes were washed and incubated with an HRP-conjugated secondary antibody (mouse #7076/rabbit #7074, Cell Signaling) at room temperature for 1 hour.

Techniques: Sonication

Journal: bioRxiv

Article Title: A pharmacological modality to sequester homomeric proteins

doi: 10.1101/2025.03.05.641597

Figure Lengend Snippet: a. Dynamic light scattering experiments following recombinant Keap1-BTB (50 μM) and EL133 (50 μM) over time at room temperature. b. WB of 20 h EL133-treated OCI-AML2 cells with and without post-treatment with excess BDX (5 μM). Full gels are available in Fig. S12. c. Dynamic light scattering experiments following recombinant Keap1-BTB (50 μM) and additional Keap1-targeting PINCHs (50 μM) over time at room temperature, and d. These PINCHs’ respective WB analysis in OCI-AML2 cells (20 h). e. Dynamic light scattering experiments following recombinant LDHA incubated with OS-47B over time at room temperature. Each LDHA bears four binding sites, and each PINCH bears two binding moieties (e.g., 5 μM LDHA= 20 μM binding sites). f. Chemical structure of LDHA-targeting PINCH OS-47B.

Article Snippet: Incubation with primary antibodies (mouse anti-BCL-6 (sc-7388, Santa Cruz, 1:500, overnight at 4 °C), mouse anti-Keap1 (sc-365626, Santa Cruz, 1:500, overnight at 4 °C), rabbit anti-c-Myc (#5605, Cell Signaling, 1:1000, overnight at 4°C), rabbit anti-p62 (ab155686, Abcam, 1:1000, overnight at 4 °C) and mouse anti-beta-actin (#3700, Cell Signaling, 1:1000, 1 h at room temperature) was performed, then the membranes were washed and incubated with an HRP-conjugated secondary antibody (mouse #7076/rabbit #7074, Cell Signaling) at room temperature for 1 hour.

Techniques: Recombinant, Incubation, Binding Assay

Journal: bioRxiv

Article Title: A pharmacological modality to sequester homomeric proteins

doi: 10.1101/2025.03.05.641597

Figure Lengend Snippet: a. WB and structure of EL230, 20h treatment in Mino cells. Full gels are available in Fig. S13. b. EL230 tested in a BCL6-positive cell line (OCI-AML2) and in a BCL6-absent cell line (HEK 293), to show inactivity in the latter. c. Monomeric versions of EL230, based on BCL6- and Keap1-ligands with linkers, in Mino cells.

Article Snippet: Incubation with primary antibodies (mouse anti-BCL-6 (sc-7388, Santa Cruz, 1:500, overnight at 4 °C), mouse anti-Keap1 (sc-365626, Santa Cruz, 1:500, overnight at 4 °C), rabbit anti-c-Myc (#5605, Cell Signaling, 1:1000, overnight at 4°C), rabbit anti-p62 (ab155686, Abcam, 1:1000, overnight at 4 °C) and mouse anti-beta-actin (#3700, Cell Signaling, 1:1000, 1 h at room temperature) was performed, then the membranes were washed and incubated with an HRP-conjugated secondary antibody (mouse #7076/rabbit #7074, Cell Signaling) at room temperature for 1 hour.

Techniques: