Journal: Acta Neuropathologica Communications

Article Title: Quantitative patterns of motor cortex proteinopathy across ALS genotypes

doi: 10.1186/s40478-020-00961-2

Figure Lengend Snippet: Summary of all cases used in this study. See supplementary data for subgroup analyses

Article Snippet: SOD1 d , StressMarq Bio , SPC-206 , Rabbit , Polyclonal , 1:1000 , –.

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: Quantitative patterns of motor cortex proteinopathy across ALS genotypes

doi: 10.1186/s40478-020-00961-2

Figure Lengend Snippet: Primary antibody clones used in this study

Article Snippet: SOD1 d , StressMarq Bio , SPC-206 , Rabbit , Polyclonal , 1:1000 , –.

Techniques: Clone Assay

Journal: Acta Neuropathologica Communications

Article Title: Quantitative patterns of motor cortex proteinopathy across ALS genotypes

doi: 10.1186/s40478-020-00961-2

Figure Lengend Snippet: Primary motor cortex and spinal cord single-IHC results

Article Snippet: SOD1 d , StressMarq Bio , SPC-206 , Rabbit , Polyclonal , 1:1000 , –.

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: Quantitative patterns of motor cortex proteinopathy across ALS genotypes

doi: 10.1186/s40478-020-00961-2

Figure Lengend Snippet: Pathology of the ALS primary motor cortex is variable both within and across the genotypic spectrum of disease. Relatively little pTDP-43 aggregation was found in a single TARDBP mutation case ( a ), but this was severe in an OPTN mutation case (d; see also supp. Figure ). Insets highlight variance of pTDP-43 morphology between genotypes. Average highest pTDP-43 deposition was seen in sporadic cases, which was statistically higher than in C9-ALS ( g ). Quantification of p62 ( b , e , h ). Levels of p62 correlated with pTDP-43 in sporadic cases but less so in C9ORF72 disease, reflective of the existence of p62-positive dipeptide repeat protein species unique to C9-ALS ( h and j ). Cortical microglial activation was highly variable between genotypes ( i ), and in some cases there was evidence of severe nodular neuronophagia surrounding layer V neurons ( f ). Grey matter CD68 correlated with the extent of pTDP-43 deposition ( k ) in both sporadic and C9ORF72 cases, but this relationship was not recapitulated using p62 and CD68 in SOD1 / FUS cases ( l ). Arrows highlight pathology, asterisks highlight Betz cells. r correlations = Pearson ( j ) or Spearman ( k , l ), results as on figure. Bars in ( i ) represent means and SEM. Best-fit lines are manually added for illustrative purposes. All scale bars = 50 μm

Article Snippet: SOD1 d , StressMarq Bio , SPC-206 , Rabbit , Polyclonal , 1:1000 , –.

Techniques: Mutagenesis, Activation Assay

Journal: Acta Neuropathologica Communications

Article Title: Quantitative patterns of motor cortex proteinopathy across ALS genotypes

doi: 10.1186/s40478-020-00961-2

Figure Lengend Snippet: Spatial and morphological distribution of proteinopathy across genotypes in the ALS primary motor cortex. Sporadic ALS exhibits NCI and oligodendroglial pathology across layers I-VI as well as the subcortical white matter ( a - d ). The distribution pattern of pTDP-43 pathology was not entirely dissimilar between sporadic and C9-ALS ( e - f ), although there was a slight preponderance towards oligodendrocyte inclusions in C9 cases (Fig. g and h). FUS mutation cases demonstrated infrequent, compact p62-positive NCI with occasional granular inclusions, as well as occasional oligodendroglial pathology (i-l). SOD1 mutation cases, by contrast, exhibited granular p62 staining confined to the middle cortical layers with infrequent NCI in layer V, without obvious glial pathology. Arrows highlight respective proteinopathy. Scale bar applicable to all panels = 50 μm

Article Snippet: SOD1 d , StressMarq Bio , SPC-206 , Rabbit , Polyclonal , 1:1000 , –.

Techniques: Mutagenesis, Staining

Journal: Redox Biology

Article Title: The loss of pancreatic islet NADPH oxidase (NOX)2 improves islet transplantation

doi: 10.1016/j.redox.2022.102419

Figure Lengend Snippet: The loss of NOX2 increases the expression of insulin and antioxidative proteins in hypoxic islets. ( A ) Representative Western blot analysis of (from top to bottom) Nrf2, β-actin, HO-1, SOD2, SOD1 and insulin from whole cell extracts of isolated hypoxic WT and Nox2 −/− islets. ( B ) Quantitative analysis of insulin expression (Fold change) (n = 5 each). Mean ± SEM. *P < 0.05 vs. WT. ( C ) Quantitative analysis of SOD1 expression (Fold change) (n = 3 each). Mean ± SEM. *P < 0.05 vs. WT. ( D ) Quantitative analysis of SOD2 expression (Fold change) (n = 3 each). Mean ± SEM. *P < 0.05 vs. WT. ( E ) Quantitative analysis of HO-1 expression (Fold change) (n = 5 each). Mean ± SEM. *P < 0.05 vs. WT. ( F ) Quantitative analysis of Nrf2 expression (Fold change) (n = 5 each). Mean ± SEM. *P < 0.05 vs. WT.

Article Snippet: The anti-SOD1 (TA321133) and anti-SOD2 (TA321189) antibodies were purchased from OriGene (Rockville, USA).

Techniques: Expressing, Western Blot, Isolation

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: This figure presents the computational prediction of the interaction between matrix metalloproteinase-9 (MMP9) binding to superoxide dismutase-3 (SOD3), achieved through molecular docking using ClusPro and AutoDock software. In visualization, MMP9 is depicted in green, and SOD3 is represented in purple. The Prodigy webserver was utilized to analyze their binding affinity, revealing a strong interaction characterized by a ΔG (kcal mol−1) value of −16.2 and a Kd (M) value of 1.4e-12, indicative of high binding affinity. Further examination identified a potential cleavage site for MMP9 on SOD3 amino acid at position 173AA with a significant score of 0.906. This suggests a probable proteolytic interaction between the two proteins. The molecular interaction and the structural remodeling of these protein complexes were visualized and assessed using Pymol and ChimeraX. Collectively, the computational analysis, binding affinity evaluation, and identification of a potential cleavage site provide insights into the protein–protein interaction between MMP9 and SOD3, highlighting their intricate molecular relationship.

Article Snippet: SOD3 was detected using an antibody from Proteintech (Cat No. 14316-1-AP).

Techniques: Binding Assay, Software

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: This figure presents a compelling visualization of the regulatory effects catalytic inactive matrix metalloproteinase-9 (MMP9) exerts on superoxide dismutase-3 (SOD3) through protein–protein interactions. (A) The representative Western blot image illustrates the protein levels of SOD3, approximately 30 kD, across various experimental conditions: a control group (CT), MMP9 cells activated by phorbol myristate acetate (PMA), MMP9 cells treated with a specific inhibitor, and cells transfected with a catalytically inactive MMP9 mutant plasmid. To ensure accuracy in protein quantification, total protein served as the loading control. The accompanying bar graph provides a detailed densitometric analysis of the Western blot bands, allowing for a quantitative comparison of SOD3 expression across the different treatment groups. This analysis represents data from six separate experiments (N = 6), ensuring a robust and reliable assessment of the experimental outcomes. (B) The co-immunoprecipitation results provide evidence of the interaction between MMP9 and SOD3 proteins. We conducted immunoprecipitation with an MMP9 antibody and subsequent immunoblotting with a SOD3 antibody on cell lysates. The detection of SOD3 in complexes with mutant MMP9 underscores a direct protein-protein interaction between MMP9 and SOD3. The accompanying bar graph offers a densitometric analysis of the SOD3 bands from the Western blot, quantitatively depicting the extent of this interaction. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test, with significance levels denoted as ** for P < 0.001 and **** for P < 0.0001. ’ns’ indicates non-significant differences. This assay was repeated thrice (N = 3) to ensure data robustness. Each point represents one sample.

Article Snippet: SOD3 was detected using an antibody from Proteintech (Cat No. 14316-1-AP).

Techniques: Protein-Protein interactions, Western Blot, Control, Transfection, Mutagenesis, Plasmid Preparation, Comparison, Expressing, Immunoprecipitation

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: The proximal ligation assay (PLA) effectively demonstrated a direct protein-protein interaction between matrix metalloproteinase-9 (MMP9) and superoxide dismutase-3 (SOD3). In this assay, we fixed the experimental cells and introduced specific antibodies targeting SOD3 and MMP9. The close proximity of these proteins was evidenced by the formation of red fluorescent dots, signifying their interaction. To confirm the assay’s specificity, we included a negative control using only one of the primary antibodies. Scale bar = 10 μm.

Article Snippet: SOD3 was detected using an antibody from Proteintech (Cat No. 14316-1-AP).

Techniques: Ligation, Negative Control

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: The Western blots and accompanying bar graphs delineate the levels of superoxide dismutase-3 (SOD3) and matrix metalloproteinase-9 (MMP9) in the culture medium of HEK293 cells. The experiment encompassed three distinct groups: a control (CT), cells transfected with an MMP9 overexpressing plasmid followed by phorbol myristate acetate (PMA) treatment to activate MMP9, and cells transfected with a catalytically inactive mutant MMP9 plasmid. The Western blot data, presented in two sections, (A) highlight MMP9 levels and (B) focus on SOD3 levels. For each sample, total protein was utilized as the loading control. We conducted statistical analyses using one-way ANOVA followed by Tukey’s post-hoc test across the groups, with each data point representing an individual sample (N = 6). Significance levels are marked as ** (P < 0.01) and **** (P < 0.0001), with ’ns’ indicating non-significant results.

Article Snippet: SOD3 was detected using an antibody from Proteintech (Cat No. 14316-1-AP).

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Mutagenesis

Journal: Canadian journal of physiology and pharmacology

Article Title: Deciphering MMP9’s dual role in regulating SOD3 through protein–protein interactions

doi: 10.1139/cjpp-2023-0256

Figure Lengend Snippet: The schematic illustrates the distinct regulatory roles of matrix metalloproteinase-9 (MMP9) on superoxide dismutase-3 (SOD3). It showcases how active MMP9 potentially enhances SOD3 levels, likely through transcriptional upregulation. In contrast, catalytically inactive MMP9 seems to reduce SOD3 levels through direct protein-protein interactions and possibly via a unique proteolytic degradation mechanism. This differential regulation by MMP9 leads to increased secretion of SOD3 from cells in its active form, while the catalytically inactive MMP9 impedes SOD3 secretion. Phorbol myristate acetate (PMA), identified as an MMP9 activator in the schematic, plays a crucial role in modulating these interactions.

Article Snippet: SOD3 was detected using an antibody from Proteintech (Cat No. 14316-1-AP).

Techniques: Protein-Protein interactions

Journal: Molecular Neurodegeneration

Article Title: Immunochemical characterization on pathological oligomers of mutant Cu/Zn-superoxide dismutase in amyotrophic lateral sclerosis

doi: 10.1186/s13024-016-0145-9

Figure Lengend Snippet: Anti-SOD1 int antibody exclusively recognizes soluble disulfide-crosslinked SOD1 oligomers in vitro. a The antibodies were tested for their specific reactivities to soluble disulfide-crosslinked oligomers (black filled bars) over Cu,Zn-SOD1(WT) S-S (open bars) and E,E-SOD1(A4V) S-S (gray filled bars) by indirect ELISA. Antisera were either affinity-purified with the corresponding peptides (w/o absorption) or first absorbed with SOD1(WT) S-S and then affinity-purified with the peptides (w/ absorption). Anti-SOD1 48–53 antibody obtained after the absorption exclusively reacted with soluble disulfide-crosslinked oligomers and called anti-SOD1 int antibody. b - d The reactivities of b anti-SOD1 int , c USOD-like, and d SEDI-like antibody were examined with indirect ELISA. Several forms of SOD1 (WT, A4V, G37R, G85R) with a distinct metallation/disulfide status, soluble disulfide-crosslinked oligomers and insoluble amyloid-like aggregates were prepared and fixed on an ELISA plate. The ELISA signal was represented as a ratio against that obtained using BSA. Three independent experiments were performed to estimate error bars (standard deviation). Fixation of equal amounts of SOD1 proteins on each well of an ELISA plate was confirmed by ELISA using polyclonal anti-SOD1 antibody (FL-154, Santa Cruz Biotechnology), which is shown in Additional file : Figure S5

Article Snippet: After six washes with TBS-T, either antibody purified in this study, polyclonal anti-human SOD1 (FL-154, Santa Cruz Biotechnology), USOD (#SPC-205, StressMarq Bioscience), or SEDI (#SPC-206, StressMarq Bioscience) antibody was added as a primary antibody (0.2 μg/mL) and incubated for an hour at room temperature, which was then followed by secondary antibody with horseradish peroxidase (goat anti-rabbit IgG, 1:1,000; Thermo Scientific) for an hour at room temperature.

Techniques: In Vitro, Indirect ELISA, Affinity Purification, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Acta biochimica et biophysica Sinica

Article Title: Vaccarin suppresses diabetic nephropathy through inhibiting the EGFR/ERK1/2 signaling pathway.

doi: 10.3724/abbs.2024141

Figure Lengend Snippet: Figure 3. VAC alleviated the renal inflammatory response and oxidative stress in T2DM mice (A) MDA contents in diabetic kidney tissues. n=6. (B) GSH-Px activity in diabetic kidney tissues. n=6. (C) Averaged fluorescence intensity of DHE fluorescence in diabetic kidney tissues. n=6. (D) Averaged fluorescence intensity of DCFH-DA fluorescence staining of diabetic kidney tissues. n=6. (E) DHE fluorescence staining or DCFH-DA fluorescence staining of diabetic kidney tissues. Scale bar: 100 μm. (F) F4/80 staining of diabetic kidney tissues. Scale bar: 50 μm, and the average fluorescence intensity of F4/80-expressing diabetic kidney tissues is shown. (G‒M) Representative blot images and quantitative analysis of phosphorylated NFκB P65, Nrf2, catalase, SOD3, SOD2 and SOD1. n=4. *P<0.05, **P<0.01, ***P<0.001 vs Ctrl. #P<0.05, ##P<0.01, ###P<0.001 vs DN.

Article Snippet: Primary antibodies against catalase, SOD1, SOD2 and SOD3 were procured from Boster Biological Technology (Wuhan, China).

Techniques: Activity Assay, Fluorescence, Staining, Expressing

Journal: Acta biochimica et biophysica Sinica

Article Title: Vaccarin suppresses diabetic nephropathy through inhibiting the EGFR/ERK1/2 signaling pathway.

doi: 10.3724/abbs.2024141

Figure Lengend Snippet: Figure 5. VAC alleviated the inflammatory response and oxidative stress in diabetic kidneys HK-2 cells were preincubated with 5 μM VAC for 12 h, followed by exposure to 35 mM HG for 48 h. (A–C) Relative mRNA levels of IL-1β, VCAM-1 and COX2. (D-F) DHE fluorescence staining or DCFH-DA fluorescence staining of HK-2 cells. Scale bar: 100 μm. (G–K) Relative mRNA levels of Nrf2, catalase, SOD3, SOD2 and SOD1. (L–R) Representative blot images and quantitative analysis of phosphorylated NFκB P65, Nrf2, catalase, SOD3, SOD2 and SOD1. *P<0.05, **P<0.01, ***P<0.001 vs NG. #P<0.05, ##P<0.01, ###P<0.001 vs HG. n=4.

Article Snippet: Primary antibodies against catalase, SOD1, SOD2 and SOD3 were procured from Boster Biological Technology (Wuhan, China).

Techniques: Fluorescence, Staining

Journal: Acta biochimica et biophysica Sinica

Article Title: Vaccarin suppresses diabetic nephropathy through inhibiting the EGFR/ERK1/2 signaling pathway.

doi: 10.3724/abbs.2024141

Figure Lengend Snippet: Figure 8. VAC ameliorated fibrosis, the inflammatory response and oxidative stress in HG-induced HK-2 cells exposed to the EGFR inhibitor AG1478 or the ERK inhibitor U0126 (A–D) Relative mRNA levels of collagen-1, TGF-β1, α-SMA and E-cadherin in HK-2 cells. (E–G) Relative mRNA levels of IL-1β, VCAM-1 and COX-2 in HK-2 cells. (H) Averaged fluorescence intensity of DHE fluorescence in HK-2 cells. (I) DHE staining was performed on HG-induced HK-2 cells. Scale bar: 100 μm. (J–P) Representative blot images and quantitative analysis of phosphorylated and total NFκB P65, Nrf2, catalase, SOD3, SOD2 and SOD1. *P<0.05, **P<0.01, ***P<0.001 vs NG. #P<0.05, ##P<0.01, ###P<0.001 vs HG. n=4.

Article Snippet: Primary antibodies against catalase, SOD1, SOD2 and SOD3 were procured from Boster Biological Technology (Wuhan, China).

Techniques: Fluorescence, Staining