Journal: Cell Death Discovery

Article Title: Regulation of cardiac ferroptosis in diabetic human heart failure: uncovering molecular pathways and key targets

doi: 10.1038/s41420-024-02044-w

Figure Lengend Snippet: Immunoblotting shows downregulation of cardiac SOD3 ( A ) and upregulation of MMP9 ( B ) in diabetic heart failure. Downregulated SOD3 and upregulated MMP9 are molecular markers of heart failure. Unpaired two-tailed t-test. * P < 0.05; *** P < 0.001. Each point represents one patient. N = 4–5. SOD3 = superoxide dismutase-3, MMP9 = matrix metalloproteinase-9, CT = control, healthy subjects, dHF = diabetic heart failure patients.

Article Snippet: The secondary antibody incubation was conducted at room temperature for 1 h. The antibodies used for the western blot detection were as follows: GPX4 (ab125066), ATF4 (10835-1-AP), GSR (18257-1-AP), NRF2 (16396-1-AP), TFR (17435-1-AP), STEAP3 (Thermo Fisher PA5-20406), DMT1 (20507-1-AP), FLC (10727-1-AP), FPN1 (26601-1-AP), APP (14-9749-82), HMOX1 (10701-1-AP), LPCAT3 (67882-1-Ig), ACOT1 (ab133948), SOD3 (14316-1-AP), MMP9 (10375-2-AP), anti-4-hydroxynonenal antibody (ab46545), caspase 3 (CST: #9662), caspase 1 (CST: #2225), caspase 8 antibody (ab25901), GSDMD (ProteinTech 66387-1-Ig), and MLKL (ProteinTech 66675-1-Ig).

Techniques: Western Blot, Two Tailed Test, Control

Journal: Cell Death Discovery

Article Title: Regulation of cardiac ferroptosis in diabetic human heart failure: uncovering molecular pathways and key targets

doi: 10.1038/s41420-024-02044-w

Figure Lengend Snippet: Immunoblotting shows downregulation of cardiac SOD3 ( A ) and upregulation of MMP9 ( B ) in diabetic heart failure. Downregulated SOD3 and upregulated MMP9 are molecular markers of heart failure. Unpaired two-tailed t-test. * P < 0.05; *** P < 0.001. Each point represents one patient. N = 4–5. SOD3 = superoxide dismutase-3, MMP9 = matrix metalloproteinase-9, CT = control, healthy subjects, dHF = diabetic heart failure patients.

Article Snippet: The secondary antibody incubation was conducted at room temperature for 1 h. The antibodies used for the western blot detection were as follows: GPX4 (ab125066), ATF4 (10835-1-AP), GSR (18257-1-AP), NRF2 (16396-1-AP), TFR (17435-1-AP), STEAP3 (Thermo Fisher PA5-20406), DMT1 (20507-1-AP), FLC (10727-1-AP), FPN1 (26601-1-AP), APP (14-9749-82), HMOX1 (10701-1-AP), LPCAT3 (67882-1-Ig), ACOT1 (ab133948), SOD3 (14316-1-AP), MMP9 (10375-2-AP), anti-4-hydroxynonenal antibody (ab46545), caspase 3 (CST: #9662), caspase 1 (CST: #2225), caspase 8 antibody (ab25901), GSDMD (ProteinTech 66387-1-Ig), and MLKL (ProteinTech 66675-1-Ig).

Techniques: Western Blot, Two Tailed Test, Control

Journal: World Journal of Gastroenterology

Article Title: N-linked glycoproteomic profiling in esophageal squamous cell carcinoma

doi: 10.3748/wjg.v28.i29.3869

Figure Lengend Snippet: Western blot validations of potential glycoprotein biomarkers. A and B: Representative Western blot results show haptoglobin (HP) (A) and procathepsin D (pCD) (B) with differential expression between esophageal squamous cell carcinoma (ESCC/T) and adjacent non-cancerous tissues (N); C: Representative Western blot results show the N-linked glycosylated fraction of HP, pCD, clusterin, superoxide dismutase 3 (SOD3), proline-arginine-rich end leucine-rich repeat protein (PRELP), and 14-3-3ζ in ESCC/T and N enriched by corresponding lectins; D: Representative Western blot results show the N-linked glycosylated fractions of clusterin, PRELP, and HP in serum of patients with ESCC/T and healthy controls. The results are representative of three independent experiments. ESCC/T: Esophageal squamous cell carcinoma; HP: Haptoglobin; pCD: Procathepsin D; SOD3: Superoxide dismutase 3; PRELP: Proline-arginine-rich end leucine-rich repeat protein.

Article Snippet: The antibodies used in this study were: Haptoglobin (1:2000, 16665-1-AP, Proteintech), cathepsin D (1:2500, ab75852, Abcam), clusterin (1:5000, 12289-1-AP, Proteintech), SOD3 (1:1500, T1799, Epitomics), PRELP (1:2000, AP6665b, Abgent), and 14-3-3-ζ (1:2500, ab85268, Abcam).

Techniques: Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Estrogen-Related Receptor γ Agonist DY131 Ameliorates Lipopolysaccharide-Induced Acute Liver Injury

doi: 10.3389/fphar.2021.626166

Figure Lengend Snippet: Primer sequence.

Article Snippet: Immunoblotting was performed using primary antibodies against ERRγ (Santa Cruz, sc-66883, 1:1,000), β-actin (Bioss, bs-0061R, 1:2,500), SOD1 (Proteintech, 67480-1-Ig, 1:5,000), SOD2 (Abclonal, A19576, 1:1,000), SOD3 (Abclonal, A6984, 1:1,000), cleaved caspase-3 (Cell Signaling Technology, 9,661, 1:1,000), and Bax (Cell Signaling Technology, 2,772, 1:1,000), followed by the incubation of peroxidase-conjugated goat anti-rabbit secondary antibody (Beyotime, A0208, 1:2,000).

Techniques: Sequencing

Journal: Frontiers in Pharmacology

Article Title: Estrogen-Related Receptor γ Agonist DY131 Ameliorates Lipopolysaccharide-Induced Acute Liver Injury

doi: 10.3389/fphar.2021.626166

Figure Lengend Snippet: DY131 ameliorated oxidative stress in LPS-treated mice. (A,B) The levels of the antioxidant GSH and oxidative stress marker MDA in livers were measured using a commercial kit (n = 10 in each group). (C) mRNA expressions of SOD1, SOD2, and SOD3 were determined by qRT-PCR (n = 10 in each group). (D) Protein levels of SOD1, SOD2, and SOD3 were detected by Western blotting (n = 8 in each group). (E) Quantitative analyses of SOD1, SOD2, and SOD3 by densitometry (n = 8 in each group). (F) Representative images of DHE staining of liver tissues in different groups (magnification ×200, scale bar: 50 μm, n = 3 in each group). (G) Quantification of the mean fluorescence intensity of DHE was analyzed by ImageJ software. Data were presented as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. the indicated group. NS, no significance.

Article Snippet: Immunoblotting was performed using primary antibodies against ERRγ (Santa Cruz, sc-66883, 1:1,000), β-actin (Bioss, bs-0061R, 1:2,500), SOD1 (Proteintech, 67480-1-Ig, 1:5,000), SOD2 (Abclonal, A19576, 1:1,000), SOD3 (Abclonal, A6984, 1:1,000), cleaved caspase-3 (Cell Signaling Technology, 9,661, 1:1,000), and Bax (Cell Signaling Technology, 2,772, 1:1,000), followed by the incubation of peroxidase-conjugated goat anti-rabbit secondary antibody (Beyotime, A0208, 1:2,000).

Techniques: Marker, Quantitative RT-PCR, Western Blot, Staining, Fluorescence, Software

Journal: International Journal of Molecular Sciences

Article Title: Anakinra Activates Superoxide Dismutase 2 to Mitigate Inflammasome Activity

doi: 10.3390/ijms22126531

Figure Lengend Snippet: Anakinra spares mitochondria from oxidative damage by inducing PGC1α and SOD2: ( a ) Scanning electron microscopy of RAW 264.7 cells exposed to anakinra for 4 h. Images were taken with the Philips XL30 field emission scanning electron microscope. Note the presence of autophagic vacuoles engulfing enlarged mitochondria (arrows in the inset) in untreated cells as opposed to intact mitochondria (arrows in the inset) in anakinra-treated cells (indicated by squares and magnified in the insets). ( b ) Expression of the peroxisome proliferator-activated receptor coactivator 1α ( Pgc1α ) by RT-PCR in alveolar macrophages exposed to anakinra in the presence or not of Aspergillus conidia. ( c – g ) RAW 264.7 cells were treated with anakinra ( c – g ) and/or Aspergillus conidia ( d , g ) for 4 h ( d , f , g ) or the indicated times ( c , e ) for the expression of SOD2 ( c , d ), SOD1 and SOD3 ( e ), catalase activity ( f ) and the expression of TXN2 ( g ). PMA: Phorbol 12-myristate 13-acetate; DPI: Diphenyleneiodonium. * p < 0.05, ** p < 0.01, two-way ANOVA, none vs. anakinra.

Article Snippet: Blots of cell lysates were incubated with antibodies against the following proteins: COPS3, Abcam, Cat. No. Ab79698; SOD1 (Novus Biologicals); SOD2, Millipore, Cat. No. 06-984; SOD2, Thermo Fisher Scientific, Cat. No. A21990, Clone 9E2BD2; SOD3: Abcam, Cat. No. Ab83108; TXN2 (Novus Biologicals); USP36, Proteintech, Cat. No. 14783-1-AP.

Techniques: Electron Microscopy, Microscopy, Expressing, Reverse Transcription Polymerase Chain Reaction, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Anakinra Activates Superoxide Dismutase 2 to Mitigate Inflammasome Activity

doi: 10.3390/ijms22126531

Figure Lengend Snippet: Anakinra promotes the interaction between SOD2, USP36, and COP9 signalosome: ( a ) Western blot analyses of RAW 264.7 cells exposed or not (none) to 10 μg/mL anakinra in the presence of cyclohexamide (CHX) and evaluated at the indicated times for SOD2 protein level. Relative densitometric evaluation was reported. ( b ) RAW 264.7 cell lysates obtained at 0, 2 and 4 h following anakinra exposure were immunoprecipitated using anti-SOD2 antibody and subsequently revealed for binding to the specific deubiquitinating protein USP36. SOD2 was used as IP control. ( c ) Cell lysates from RAW 264.7 cells treated with anakinra were immunoprecipitated using COPS3 antibody and were revealed for binding to both USP36 and SOD2. COPS3 was used as IP control. WCL: whole cell lysate; IP: immunoprecipitation.

Article Snippet: Blots of cell lysates were incubated with antibodies against the following proteins: COPS3, Abcam, Cat. No. Ab79698; SOD1 (Novus Biologicals); SOD2, Millipore, Cat. No. 06-984; SOD2, Thermo Fisher Scientific, Cat. No. A21990, Clone 9E2BD2; SOD3: Abcam, Cat. No. Ab83108; TXN2 (Novus Biologicals); USP36, Proteintech, Cat. No. 14783-1-AP.

Techniques: Western Blot, Immunoprecipitation, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Anakinra Activates Superoxide Dismutase 2 to Mitigate Inflammasome Activity

doi: 10.3390/ijms22126531

Figure Lengend Snippet: Anakinra and SOD2 ameliorates the inflammatory pathology in vivo: ( a ) C57BL/6 mice were treated with rotenone in the presence or absence of anakinra and evaluated for lung histopathology. ( b – d ) C57BL/6 mice were infected intranasally with live A. fumigatus conidia and treated with Si Sod2 or equivalent doses of nonspecific, scrambled SiRNA in a volume of 20 µl of duplex buffer before evaluation of fungal growth (log10 cfu mean ± SEM) ( b ), lung histopathology ( c ) and cytokine quantification ( d ) at 3 dpi. Scale bar, 500 μm. **, p < 0.01; ***, p < 0.001, one-way ANOVA.

Article Snippet: Blots of cell lysates were incubated with antibodies against the following proteins: COPS3, Abcam, Cat. No. Ab79698; SOD1 (Novus Biologicals); SOD2, Millipore, Cat. No. 06-984; SOD2, Thermo Fisher Scientific, Cat. No. A21990, Clone 9E2BD2; SOD3: Abcam, Cat. No. Ab83108; TXN2 (Novus Biologicals); USP36, Proteintech, Cat. No. 14783-1-AP.

Techniques: In Vivo, Histopathology, Infection

Journal: International Journal of Molecular Sciences

Article Title: Anakinra Activates Superoxide Dismutase 2 to Mitigate Inflammasome Activity

doi: 10.3390/ijms22126531

Figure Lengend Snippet: Anakinra increased the lung expression of SOD2 in vivo: C57BL/6 ( a , b ), p47 phox−/− ( a ) and Cftr F508del ( b ) mice were infected with live Aspergillus conidia and treated with 10 mg/kg anakinra intraperitoneally for 6 consecutive days before analysis of SOD2 protein expression by immunofluorescence. Scale bar, 100 μm. Naïve: untreated mice, None: mice infected with Aspergillus conidia; anakinra: mice infected with Aspergillus conidia and treated with anakinra.

Article Snippet: Blots of cell lysates were incubated with antibodies against the following proteins: COPS3, Abcam, Cat. No. Ab79698; SOD1 (Novus Biologicals); SOD2, Millipore, Cat. No. 06-984; SOD2, Thermo Fisher Scientific, Cat. No. A21990, Clone 9E2BD2; SOD3: Abcam, Cat. No. Ab83108; TXN2 (Novus Biologicals); USP36, Proteintech, Cat. No. 14783-1-AP.

Techniques: Expressing, In Vivo, Infection, Immunofluorescence

Journal: Journal of Molecular Neuroscience

Article Title: Proteomic Profiling and Therapeutic Targeting of Oxidative Stress in Autoimmune Encephalitis

doi: 10.1007/s12031-025-02332-9

Figure Lengend Snippet: Oxidative stress-related models for diagnosing patients with AE. A – C NaiveBayes, RandomForest, and SVM for selection of oxidative stress-related variables for differentiating patients with AE from controls. D – F ROCs of NaiveBayes, RandomForest, and SVM models in diagnosing patients with AE in training, test, and total sets. G Venn diagram of the key oxidative stress-related variables (ALB, APOE, GPX3, and SOD3) through intersecting the three models. H ROCs of ALB, APOE, GPX3, and SOD3 in diagnosing patients with AE. I Correlation analysis of ALB, APOE, GPX3, and SOD3 with oxidative stress score

Article Snippet: After treatment with blocking buffer, the sections were incubated with primary antibodies of ALB (1:50; 16,475–1-AP; Proteintech), APOE (1:50; 18,254–1-AP; Proteintech), GPX3 (1:200; 3947–1-AP; Proteintech), SOD3 (1:50; ab80946; Abcam), GFAP (1:50; 16,825–1-AP; Proteintech), and Iba-1 (1:50; 66,827–1-Ig; Proteintech) at 4 °C overnight, with subsequent fluorochrome-conjugated secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI (C1002; Beyotime).

Techniques: Selection

Journal: Journal of Molecular Neuroscience

Article Title: Proteomic Profiling and Therapeutic Targeting of Oxidative Stress in Autoimmune Encephalitis

doi: 10.1007/s12031-025-02332-9

Figure Lengend Snippet: Antioxidant ALB, APOE, GPX3, and SOD3 are lowly expressed both in the serum of AE patients and CNS of EAE C57BL/6 mice. A – E Western blot of ALB, APOE, GPX3, and SOD3 in spinal cord tissues of control and EAE mice. F Transcriptional regulatory network. Triangles represent predicted transcription factors, and the circle represents the four target genes (red, high expression in autoimmune encephalitis, and green, low expression in autoimmune encephalitis). Dashed lines represent the predicted transcriptional regulatory relationships in the two databases, and solid lines represent the predicted transcriptional regulatory relationships in the three databases. G Quantification of the fractions of immune cells in patients with AE and controls. H – K Correlations between ALB, APOE, GPX3 and SOD3, and immune cells; **** p < 0.0001

Article Snippet: After treatment with blocking buffer, the sections were incubated with primary antibodies of ALB (1:50; 16,475–1-AP; Proteintech), APOE (1:50; 18,254–1-AP; Proteintech), GPX3 (1:200; 3947–1-AP; Proteintech), SOD3 (1:50; ab80946; Abcam), GFAP (1:50; 16,825–1-AP; Proteintech), and Iba-1 (1:50; 66,827–1-Ig; Proteintech) at 4 °C overnight, with subsequent fluorochrome-conjugated secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI (C1002; Beyotime).

Techniques: Western Blot, Control, Expressing

Journal: Journal of Molecular Neuroscience

Article Title: Proteomic Profiling and Therapeutic Targeting of Oxidative Stress in Autoimmune Encephalitis

doi: 10.1007/s12031-025-02332-9

Figure Lengend Snippet: Anti-oxidative stress by NAC elevates the expression of antioxidant ALB, APOE, GPX3, and SOD3 in the CNS of EAE C57BL/6 mice. A – H Immunofluorescence of A and B ALB, C and D APOE, E and F GPX3, and G and H SOD3 in spinal cord tissues of control mice, EAE mice, and NAC-administrated EAE mice. Scale bars, 50 µm; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: After treatment with blocking buffer, the sections were incubated with primary antibodies of ALB (1:50; 16,475–1-AP; Proteintech), APOE (1:50; 18,254–1-AP; Proteintech), GPX3 (1:200; 3947–1-AP; Proteintech), SOD3 (1:50; ab80946; Abcam), GFAP (1:50; 16,825–1-AP; Proteintech), and Iba-1 (1:50; 66,827–1-Ig; Proteintech) at 4 °C overnight, with subsequent fluorochrome-conjugated secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI (C1002; Beyotime).

Techniques: Expressing, Immunofluorescence, Control

Journal: Journal of Molecular Neuroscience

Article Title: Proteomic Profiling and Therapeutic Targeting of Oxidative Stress in Autoimmune Encephalitis

doi: 10.1007/s12031-025-02332-9

Figure Lengend Snippet: NAC treatment attenuates oxidative stress, tissue-resident CD4 + and CD8 + T cells and neuroinflammation in EAE SJL mice. A and B Clinical score of EAE severity and latency to fall of control, EAE, and NAC-treated EAE mice. C – E Detection of C MDA content, D SOD activity, and E GSH content in spinal cord tissues of the above mice. F – J Western blot of F and G ALB, F and H APOE, F and I GPX3, and F and J SOD3 in spinal cord tissues of the above mice. K – N Flow cytometry of the proportions of tissue-resident K and L CD4 + and M and N CD8. + T cells in spinal cord tissues of the above mice. O – Q ELISA of the levels of O IFN-γ, P TNF-α and Q IL-1β in spinal cord tissues of the above mice; *** p < 0.001; **** p < 0.0001

Article Snippet: After treatment with blocking buffer, the sections were incubated with primary antibodies of ALB (1:50; 16,475–1-AP; Proteintech), APOE (1:50; 18,254–1-AP; Proteintech), GPX3 (1:200; 3947–1-AP; Proteintech), SOD3 (1:50; ab80946; Abcam), GFAP (1:50; 16,825–1-AP; Proteintech), and Iba-1 (1:50; 66,827–1-Ig; Proteintech) at 4 °C overnight, with subsequent fluorochrome-conjugated secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI (C1002; Beyotime).

Techniques: Control, Activity Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Caveolin-1 stabilizes ATP7A, a copper transporter for extracellular SOD, in vascular tissue to maintain endothelial function

doi: 10.1152/ajpcell.00151.2020

Figure Lengend Snippet: Protein expression of the copper transporter ATP7A is decreased in blood vessels from caveolin-1 knockout (KO) mice (Cav-1−/−). A: relative protein expression of ATP7A and Atox1 in aortas from Cav-1 wild-type (WT) and Cav-1−/− mice was determined by Western blotting with antibodies specific to respective proteins. Densitometric analysis is shown (right, n = 5). B: ATP7A mRNA expression in aortas from Cav-1 WT and Cav-1−/− mice was determined by real-time quantitative RT-PCR (n = 3). C: protein expression for ATP7A and Atox1 in mouse fibroblasts isolated from Cav-1 WT and Cav-1−/− mice (n = 3). D: Cav-1 WT and Cav-1−/− mouse fibroblasts mice were transfected with Cav-1 WT plasmid. Lysates were used to measure ATP7A, Cav-1, and actin protein expression. (n = 3). E: superoxide dismutase (SOD3)-specific activity in conditional medium of Cav-1 WT plasmid transfected Cav-1−/− mouse fibroblast cells. Cav-1 WT and Cav-1−/− mouse fibroblast cells were transfected with Cav-1-WT plasmid. The specific activity of SOD3 was determined by the ratio of activity to relative amount of protein in culture conditional medium. Results are presented as means ± SE. *P < 0.05, NS, not significant. ATP7a, Menkes ATPase, copper-transporting P-type ATPase.

Article Snippet: The following primary antibodies were used: anti-Atox1 (homemade) ( 23 ), anti-SOD3 (homemade) ( 13 ), anti-SOD1 (ab16831; Abcam), anti-ATP7A (B8162; LifeSpan Biosciences), anti-actin (SC-1616; Santa Cruz Biotechnology), anti-ubiquitin (SC-8017; Santa Cruz Biotechnology), anti-Cav-1 (610407 or 610060; BD Transduction), anti-paxillin (612405; BD Transduction), anti-flotillin-1 (610820; BD Transduction), anti-heparan sulfate (MAB2040; Millipore), anti-fibulin-5 (12188; Proteintech) or anti-collagen type-1 (Proteintech, 14695).

Techniques: Expressing, Knock-Out, Western Blot, Quantitative RT-PCR, Isolation, Transfection, Plasmid Preparation, Activity Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Caveolin-1 stabilizes ATP7A, a copper transporter for extracellular SOD, in vascular tissue to maintain endothelial function

doi: 10.1152/ajpcell.00151.2020

Figure Lengend Snippet: Caveolin-1 (Cav-1) is required for ATP7A protein stabilization and prevents proteosomal degradation. A: Cav-1 wild-type (WT) and Cav-1−/− [knockout (KO)] mouse fibroblasts were lysed and immunoprecipitated (IP) with anti-ATP7A, followed by immunoblotting (IB) with an anti-ubiquitin antibody. Right: averaged data for ATP7A ubiquitination (n = 3). B: mouse fibroblast cells were incubated with an inhibitor of the proteasome MG132 (20 μmol/L) for 24 h or an inhibitor of the lysosome chloroquine (100 μmol/L) for 24 h, and the protein expression of ATP7A was determined by Western blot (n = 3). C: membrane fractionation of mouse aorta. Equivolume fractions isolated from the top (fraction 1) to the bottom (fraction 13) were immunoblotted with antibodies as indicated. D: IP of Cav-1 using an anti-Cav-1 antibody in aortic lysates, followed by IB with an ATP7A antibody (n = 3). Results are presented as means ± SE. *P < 0.05. ATP7a, Menkes ATPase, copper-transporting P-type ATPase.

Article Snippet: The following primary antibodies were used: anti-Atox1 (homemade) ( 23 ), anti-SOD3 (homemade) ( 13 ), anti-SOD1 (ab16831; Abcam), anti-ATP7A (B8162; LifeSpan Biosciences), anti-actin (SC-1616; Santa Cruz Biotechnology), anti-ubiquitin (SC-8017; Santa Cruz Biotechnology), anti-Cav-1 (610407 or 610060; BD Transduction), anti-paxillin (612405; BD Transduction), anti-flotillin-1 (610820; BD Transduction), anti-heparan sulfate (MAB2040; Millipore), anti-fibulin-5 (12188; Proteintech) or anti-collagen type-1 (Proteintech, 14695).

Techniques: Knock-Out, Immunoprecipitation, Western Blot, Incubation, Expressing, Fractionation, Isolation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Caveolin-1 stabilizes ATP7A, a copper transporter for extracellular SOD, in vascular tissue to maintain endothelial function

doi: 10.1152/ajpcell.00151.2020

Figure Lengend Snippet: Transgenic mice overexpressing Menkes ATPase, copper-transporting P-type ATPase (ATP7A; ATP7A Tg) rescued superoxide dismutase (SOD3) activity and endothelium-dependent relaxation in caveolin-1 knockout (KO) (Cav-1−/−) mice. A: protein levels of ATP7A, SOD3, SOD1, Cav-1, and actin in aortas from Cav-1 wild-type (WT) or Cav-1−/− double Tg mice overexpressing ATP7A were measured (n = 3). B: specific activity of SOD3 and SOD1 in tissue homogenates were assayed as described in Fig. 1. C: endothelium-dependent or -independent relaxation of mesenteric resistance arteries from Cav-1 WT or Cav-1−/− double Tg mice overexpressing ATP7A. Vasorelaxation was evoked by acetylcholine (ACh) and sodium nitroprusside (SNP) after preconstriction with phenylephrine (n = 6). D: membrane fractionation of mouse aorta of Cav-1 WT, Cav-1−/−, and ATP7A-Tg/Cav-1−/− mice. Equal amounts of caveolin-enriched lipid raft fractions (caveolae/lipid rafts; fraction 4 to 5) and non-caveolae/lipid rafts; fractions 10 to 13) from a pool of 4 mouse aortas were immunoblotted with antibodies as indicated. Results are presented as means ± SE. *P < 0.05. ATP7a, Menkes ATPase, copper-transporting P-type ATPase.

Article Snippet: The following primary antibodies were used: anti-Atox1 (homemade) ( 23 ), anti-SOD3 (homemade) ( 13 ), anti-SOD1 (ab16831; Abcam), anti-ATP7A (B8162; LifeSpan Biosciences), anti-actin (SC-1616; Santa Cruz Biotechnology), anti-ubiquitin (SC-8017; Santa Cruz Biotechnology), anti-Cav-1 (610407 or 610060; BD Transduction), anti-paxillin (612405; BD Transduction), anti-flotillin-1 (610820; BD Transduction), anti-heparan sulfate (MAB2040; Millipore), anti-fibulin-5 (12188; Proteintech) or anti-collagen type-1 (Proteintech, 14695).

Techniques: Transgenic Assay, Activity Assay, Knock-Out, Fractionation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Caveolin-1 stabilizes ATP7A, a copper transporter for extracellular SOD, in vascular tissue to maintain endothelial function

doi: 10.1152/ajpcell.00151.2020

Figure Lengend Snippet: Proposed working model showing how caveolin-1 (Cav-1) is required for stabilizing protein expression of the copper transporter ATP7A and enabling copper delivery to extracellular superoxide dismutase 3 (SOD3) in vascular tissue to enable its full activity and protection of endothelial function against oxidative stress. ATP7A, Menkes ATPase, copper (Cu)-transporting P-type ATPase; Ub, ubiquitin.

Article Snippet: The following primary antibodies were used: anti-Atox1 (homemade) ( 23 ), anti-SOD3 (homemade) ( 13 ), anti-SOD1 (ab16831; Abcam), anti-ATP7A (B8162; LifeSpan Biosciences), anti-actin (SC-1616; Santa Cruz Biotechnology), anti-ubiquitin (SC-8017; Santa Cruz Biotechnology), anti-Cav-1 (610407 or 610060; BD Transduction), anti-paxillin (612405; BD Transduction), anti-flotillin-1 (610820; BD Transduction), anti-heparan sulfate (MAB2040; Millipore), anti-fibulin-5 (12188; Proteintech) or anti-collagen type-1 (Proteintech, 14695).

Techniques: Expressing, Activity Assay