anti p enos  (Cell Signaling Technology Inc)

Journal: Journal of Lipid Research

Article Title: S-nitrosylation contributes to ER stress and aggresome formation in secosterol-B-mediated endothelial dysfunction

doi: 10.1016/j.jlr.2026.101017

Figure Lengend Snippet: SEC-B induces NSS through increased intracellular NO and RNS and depletion of the antioxidant GSH. Quantitative analysis of RNS (A), GSH (B), and NO (C) levels in HUVECs treated with 10 μM SEC-B. Data are expressed as the mean ± SD of the values obtained from at least three independent experiments.∗ P < 0.05, ∗∗ P < 0.01 versus untreated cells. D: Confocal microscopy of 4,5-diaminofluorescein diacetate-stained HUVECs after 24 h of treatment with 10 μM SEC-B. The scale bar represents 20 μm. E and F: Western immunoblotting analysis and quantitation of iNOS and eNOS expression levels in HUVECs treated with 10 μM SEC-B. Data are reported in the graph as fold change versus untreated cells. Data are expressed as the mean ± SD of the values obtained from at least four independent experiments. ∗ P < 0.05 versus untreated cells.

Article Snippet: Blots were probed with the following antibodies: anti-spliced X-box binding protein 1 (XBP-1s) (E9V3E, #40435), ATF6 (D4Z8V, #65880), BiP/GRP78 (C50B12, #3177), and protein disulfide isomerase (PDI) (#2446) from Cell Signaling Technology; anti-endothelial NOS (eNOS) (A1548) from ABclonal; and anti-NOS2 (N-20, sc-651) from Santa Cruz Biotechnology.

Techniques: Confocal Microscopy, Staining, Western Blot, Quantitation Assay, Expressing

Journal: Renal Failure

Article Title: ADAMTS13 ameliorates diabetic nephropathy by Nrf2/GPX4/eNOS signaling pathway

doi: 10.1080/0886022X.2026.2646089

Figure Lengend Snippet: ADAMTS13 activated Nrf2/GPX4/eNOS pathway, inhibited aortic oxidative stress level and ameliorated endothelial dysfunction in DN mice. (A) Representative images and quantification of Nrf2, SOD1, and SOD2 by western blot analysis. (B) Representative images and quantification of GPX4, eNOS, and p-eNOS by western blot analysis. Nrf2: nuclear factor erythroid 2-related factor 2; SOD: superoxide dismutase; GPX4: glutathione peroxidase 4; eNOS: endothelial nitric oxide synthase. Results were presented as mean ± SEM. N = 3, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: Equal amounts of protein were loaded on 8–12% sodium dodecyl sulfate-polyacrylamide gels, transferred to polyvinylidene fluoride membrane (PVDF) (Thermo Fisher Scientific, Waltham, MA), and blocked with 5% skim milk powder for 2 h. Membranes were incubated overnight at 4 °C with the following primary antibodies: Beclin-1 (A7353, ABclonal, Wuhan, China); LC3II/I (#4108, 14/16 kDa, CST, Danvers, MA); Nrf2 (ab31163, Abcam, Cambridge, UK); SOD1 (A12537, ABclonal, Wuhan, China); SOD2 (A21805, ABclonal, Wuhan, China); GPX4 (A1933, ABclonal, Wuhan, China); eNOS (#32027, CST, Danvers, MA); p-eNOS (Ser 1177) (AF5812, Beyotime, Shanghai, China); β-actin (AF5002, Beyotime, Shanghai, China); GAPDH (AC001, ABclonal, Wuhan, China); Histone H3 (A2348, ABclonal, Wuhan, China).

Techniques: Western Blot

Journal: Renal Failure

Article Title: ADAMTS13 ameliorates diabetic nephropathy by Nrf2/GPX4/eNOS signaling pathway

doi: 10.1080/0886022X.2026.2646089

Figure Lengend Snippet: ADAMTS13 promoted NO production and alleviated oxidative stress by Nrf2/GPX4/eNOS signaling pathway in high glucose-induced HGECs. (A, E, and I) Representative images of DAF-FM DA, DHE, and MitoSOX staining were used to assess the production of NO, the cytoplasmic and the mitochondrial ROS generation in HGECs. Scale bar: 100 μm. (B, F, and J) The fluorescence intensity of DAF-FM DA. (C, G, and K) The fluorescence intensity of DHE. (D, H, and L) The fluorescence intensity of MitoSOX. HGECs: human glomerular endothelial cells. Results were presented as mean ± SEM. N = 5, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: Equal amounts of protein were loaded on 8–12% sodium dodecyl sulfate-polyacrylamide gels, transferred to polyvinylidene fluoride membrane (PVDF) (Thermo Fisher Scientific, Waltham, MA), and blocked with 5% skim milk powder for 2 h. Membranes were incubated overnight at 4 °C with the following primary antibodies: Beclin-1 (A7353, ABclonal, Wuhan, China); LC3II/I (#4108, 14/16 kDa, CST, Danvers, MA); Nrf2 (ab31163, Abcam, Cambridge, UK); SOD1 (A12537, ABclonal, Wuhan, China); SOD2 (A21805, ABclonal, Wuhan, China); GPX4 (A1933, ABclonal, Wuhan, China); eNOS (#32027, CST, Danvers, MA); p-eNOS (Ser 1177) (AF5812, Beyotime, Shanghai, China); β-actin (AF5002, Beyotime, Shanghai, China); GAPDH (AC001, ABclonal, Wuhan, China); Histone H3 (A2348, ABclonal, Wuhan, China).

Techniques: Staining, Fluorescence

Journal: bioRxiv

Article Title: DOT1L-AF10–mediated H3K79me3 promotes NF-κB p65–dependent inflammatory activation in endothelial cells

doi: 10.64898/2026.03.20.713137

Figure Lengend Snippet: (A-E) Immunoblot analyses of EA.hy926 treated with TNF-α (10 ng/ml, 24 h) in the presence or absence of the DOT1L inhibitor SYC-522 (5 µM, 24 h), showing the expression of H3K79me3 (n = 4; A), eNOS (n = 5; B), ICAM1 (n = 3; C), Phospho-NF-κB p65 (n = 4), total NF-κB p65 (n = 3), Phospho-IκBα (n = 3), total IκBα (n = 3; D), Phospho-IKKα/β (n = 3), and total IKKβ (n = 3; E). (F) Analyzing the transcript levels of NF-κB p65 in HUVEC EA.hy926 treated with TNF-α (10 ng/ml, 24 h) in the presence or absence of DOT1L siRNA (siDOT1L, 40 nM). (n = 3) (G-I) Immunoblot analyses of rat aortic rings treated with TNF-α (10 ng/ml, 24 h) ex vivo in the presence or absence of the DOT1L inhibitor SYC-522 (5 µM, 24 h), showing the expression of eNOS (n = 3; G), VCAM1 (n = 3; H), and total NF-κB p65 (I, n = 3). (J) En face preparations were double stained with anti–VE-cadherin (green, EC marker) and an anti-NF-kB p65 antibody (red, C) in TNF-α and/or DOT1L inhibitor (SYC-522) treated aortic tissue. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: For tissue sections, permeabilization was performed using 0.5% Triton X-100, followed by blocking with 10% goat serum supplemented with anti-mouse IgG (1:1000) for 1 h. Cells and tissue sections were incubated overnight at 4°C with the following primary antibodies: DOT1L Rabbit mAb (1:500; #90878), H3K79me3 Rabbit mAb (1:500; #74073), eNOS Rabbit mAb (1:100; #32027), ICAM1 Rabbit pAb (1:500; #4915), NF-κB p65 Rabbit mAb (1:800; #8242, Cell Signaling Technology, Danvers, USA), H3K79me3 Rabbit pAb (1:50; #PA5-96121), AF10 Rabbit pAb (1:500; #BS-3696R), AF17 Rabbit pAb (1:1000; #A302-198A, Invitrogen), and CD144 Mouse mAb (1:25; #SC-9989, Santa Cruz Biotechnology, Dallas, USA).

Techniques: Western Blot, Expressing, Ex Vivo, Staining, Marker

Journal: bioRxiv

Article Title: DOT1L-AF10–mediated H3K79me3 promotes NF-κB p65–dependent inflammatory activation in endothelial cells

doi: 10.64898/2026.03.20.713137

Figure Lengend Snippet: (A-E) Immunoblot analyses of EA.hy926 treated with TNF-α (10 ng/ml, 24 h) in the presence or absence of DOT1L siRNA (siDOT1L, 40 nM), showing the expression of DOT1L (n = 4; A), H3K79me3 (n = 3; B), eNOS (n = 4; C), ICAM1 (n = 3; D), and NF-κB p65 (n = 3; E). (F) Immunofluorescence staining of EA.hy926 treated with TNF-α (10 ng/ml, 24 h) in the presence or absence of GFP-FBXL10 plasmid (pFBXL10, 250 ng/ml), showing NF-κB p65 expression (red). Nuclei are stained with DAPI (blue). Fluorescence intensities (AU) in individual EC (dots) from three independent experiments are indicated together with the mean. A total of ≥ 55 EC were analyzed per condition. Magnification: x63; Scale: 20 µm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: For tissue sections, permeabilization was performed using 0.5% Triton X-100, followed by blocking with 10% goat serum supplemented with anti-mouse IgG (1:1000) for 1 h. Cells and tissue sections were incubated overnight at 4°C with the following primary antibodies: DOT1L Rabbit mAb (1:500; #90878), H3K79me3 Rabbit mAb (1:500; #74073), eNOS Rabbit mAb (1:100; #32027), ICAM1 Rabbit pAb (1:500; #4915), NF-κB p65 Rabbit mAb (1:800; #8242, Cell Signaling Technology, Danvers, USA), H3K79me3 Rabbit pAb (1:50; #PA5-96121), AF10 Rabbit pAb (1:500; #BS-3696R), AF17 Rabbit pAb (1:1000; #A302-198A, Invitrogen), and CD144 Mouse mAb (1:25; #SC-9989, Santa Cruz Biotechnology, Dallas, USA).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Plasmid Preparation, Fluorescence

Journal: bioRxiv

Article Title: DOT1L-AF10–mediated H3K79me3 promotes NF-κB p65–dependent inflammatory activation in endothelial cells

doi: 10.64898/2026.03.20.713137

Figure Lengend Snippet: (A-C) Immunofluorescence staining of EA.hy926 exposed to D-Flow (4 h; n = 3), showing the expression of H3K79me3 (A), eNOS (B), and NF-κB p65 (C). Nuclei are stained with DAPI (blue), and target signals corresponding to each respective protein are in red. Fluorescence intensities (AU) in individual EC (dots) from three independent experiments are indicated together with the mean. A total of ≥ 60 EC were analyzed per condition. Magnification: x20; Scale: 100 µm. (D) Monocyte adhesion assay: DiI-labeled THP-1 were incubated (30 min) with flow-exposed EA.hy926 (4 h; n = 4) in the presence or absence of SYC-522 (5 µM). Adherent monocytes (red dots) were counted in regions exposed to S-Flow and D-Flow. Magnification: x10; Scale: 100 µm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: For tissue sections, permeabilization was performed using 0.5% Triton X-100, followed by blocking with 10% goat serum supplemented with anti-mouse IgG (1:1000) for 1 h. Cells and tissue sections were incubated overnight at 4°C with the following primary antibodies: DOT1L Rabbit mAb (1:500; #90878), H3K79me3 Rabbit mAb (1:500; #74073), eNOS Rabbit mAb (1:100; #32027), ICAM1 Rabbit pAb (1:500; #4915), NF-κB p65 Rabbit mAb (1:800; #8242, Cell Signaling Technology, Danvers, USA), H3K79me3 Rabbit pAb (1:50; #PA5-96121), AF10 Rabbit pAb (1:500; #BS-3696R), AF17 Rabbit pAb (1:1000; #A302-198A, Invitrogen), and CD144 Mouse mAb (1:25; #SC-9989, Santa Cruz Biotechnology, Dallas, USA).

Techniques: Immunofluorescence, Staining, Expressing, Fluorescence, Cell Adhesion Assay, Labeling, Incubation