Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: Generation of motor neuron models of ALS in which misfolded and aggregated hSOD1 are intracellularly deposited. NSC-34 cells were transiently transfected with expression vectors encoding human wild-type superoxide dismutase 1 (hSOD1 WT ) or the ALS-causing hSOD1 mutants (G93A, G37R, D90A, and G85R) tagged with green fluorescent protein (GFP), treated with all trans -retinoic acid, and cultured for 72 h. ( a ) Western blots of hSOD1 in the Nonidet P-40 (NP-40) soluble and insoluble factions from NSC-34 cells, β-tubulin was used as a loading control. No Vec., untransfected cells; ( b ) florescence microscopy of cells transfected with hSOD1 WT GFP or the ALS-linked hSOD1 mutant GFP. Arrows indicate protein aggregations containing hSOD1 GFP. Scale bar = 10 µm; ( c ) enzyme-linked immunosorbent assay signals of (black) EDI-positive and (gray) C4F6-positive misfolded SOD1 in the NP-40 soluble fraction. All data are shown as the mean ± SD, n = 3 in each transfection.

Article Snippet: As capture antibodies, two sets of antibodies specific for misfolded SOD1, EDI (0.5 µg/mL, SPC-206D StressMarq Biosciences, Victoria, BC, Canada) [ ] and C4F6 (×2000, MM-0070-2-P, Médimabs, Montreal, QC, Canada) [ ], were coated onto 96-well plates (Nunc-Immuno Module plate, Nunc, Roskilde, Denmark) at 4 °C for 18 h. The wells of the plates were washed three times with PBS (pH 7.4) containing 0.05% ( v / v ) Tween 20 and 5 mM EDTA, and then blocked with PBS (pH 7.4) containing 3% ( w / v ) bovine serum albumin and 5 mM EDTA at room temperature for 1 h. The NP-40 soluble faction (40 µg protein/well) or conditioned medium (40 µg protein/well) was added to the wells and incubated at room temperature for 1 h. To determine the absolute level of misfolded SOD1, metal-free hSOD1 with disulfide bonds (apo-SOD1 S-S ) was used as a standard to create a calibration curve.

Techniques: Transfection, Expressing, Cell Culture, Western Blot, Microscopy, Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: Decrease in the total level of extracellular hSOD1 mutants, but not murine SOD1, in the conditioned medium. The NSC-34 cells were transiently transfected with human wild-type superoxide dismutase 1 (hSOD1 WT ) and the ALS-causing hSOD1 mutants (G93A, G37R, D90A, and G85R) fused with green fluorescent protein (GFP). After 72 h of transfection, the conditioned medium from the cells was collected; ( a ) Western blots of hSOD1-GFP and endogenous murine SOD1 in the medium, β-tubulin was used as a marker for intracellular non-secreted protein to confirm whether cell lysis or debris could be contaminated in the conditioned medium. The amount of protein loaded for sodium dodecyl sulfate polyacrylamide gel electrophoresis was validated by Ponceau S staining; ( b , c ) quantification of the absolute total protein level of extracellular ( b ) hSOD1 and ( c ) murine SOD1. Data are shown as the mean ± SD, n = 3 in each transfection. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test. ** p < 0.01 vs. hSOD1 WT .

Article Snippet: As capture antibodies, two sets of antibodies specific for misfolded SOD1, EDI (0.5 µg/mL, SPC-206D StressMarq Biosciences, Victoria, BC, Canada) [ ] and C4F6 (×2000, MM-0070-2-P, Médimabs, Montreal, QC, Canada) [ ], were coated onto 96-well plates (Nunc-Immuno Module plate, Nunc, Roskilde, Denmark) at 4 °C for 18 h. The wells of the plates were washed three times with PBS (pH 7.4) containing 0.05% ( v / v ) Tween 20 and 5 mM EDTA, and then blocked with PBS (pH 7.4) containing 3% ( w / v ) bovine serum albumin and 5 mM EDTA at room temperature for 1 h. The NP-40 soluble faction (40 µg protein/well) or conditioned medium (40 µg protein/well) was added to the wells and incubated at room temperature for 1 h. To determine the absolute level of misfolded SOD1, metal-free hSOD1 with disulfide bonds (apo-SOD1 S-S ) was used as a standard to create a calibration curve.

Techniques: Transfection, Western Blot, Marker, Lysis, Polyacrylamide Gel Electrophoresis, Staining

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: The population of extracellular Cu/Zn-deficient hSOD1 was increased in the conditioned medium. The conditioned medium from the NSC-34 cells transfected with human wild-type superoxide dismutase 1 (hSOD1 WT )-green fluorescence protein (GFP), the ALS-linked hSOD1-GFP, or GFP alone, was treated with 100 mM iodoacetamide to block artificial oxidation of the thiol group of proteins. Then, extracellular human and murine SOD1 enzymatic activity in the medium was measured by a SOD Assay Kit-WST. The level of active SOD1 in the medium was calculated based on a calibration curve that was generated from a Cu/Zn SOD1 standard. The extracellular level of active ( a ) hSOD1 and ( b ) endogenous murine SOD1 in the medium; ( c ) the medium was treated with 1 mM CuSO 4 , 1 mM ZnSO 4 , or both, for 24 h, and active hSOD1 levels in the medium were quantified using a SOD Assay Kit-WST. All data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test, n = 3 for each treatment or for each transfection. ** p < 0.01 vs. H 2 O-added medium that corresponds to the same transfection.

Article Snippet: As capture antibodies, two sets of antibodies specific for misfolded SOD1, EDI (0.5 µg/mL, SPC-206D StressMarq Biosciences, Victoria, BC, Canada) [ ] and C4F6 (×2000, MM-0070-2-P, Médimabs, Montreal, QC, Canada) [ ], were coated onto 96-well plates (Nunc-Immuno Module plate, Nunc, Roskilde, Denmark) at 4 °C for 18 h. The wells of the plates were washed three times with PBS (pH 7.4) containing 0.05% ( v / v ) Tween 20 and 5 mM EDTA, and then blocked with PBS (pH 7.4) containing 3% ( w / v ) bovine serum albumin and 5 mM EDTA at room temperature for 1 h. The NP-40 soluble faction (40 µg protein/well) or conditioned medium (40 µg protein/well) was added to the wells and incubated at room temperature for 1 h. To determine the absolute level of misfolded SOD1, metal-free hSOD1 with disulfide bonds (apo-SOD1 S-S ) was used as a standard to create a calibration curve.

Techniques: Transfection, Fluorescence, Blocking Assay, Activity Assay, Generated

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: Extracellular misfolded human superoxide dismutase 1 (hSOD1) presents as a metal-free, disulfide oxidized form (apo-SOD1 S-S ). Enzyme-linked immunosorbent assay signals of ( a ) EDI-positive misfolded SOD1; ( b ) C4F6-positive misfolded SOD1; ( c ) total hSOD1, in the conditioned medium treated with or without both 1 mM CuSO 4 and 1 mM ZnSO 4 for 24 h. Data are expressed as the mean ± SD, n = 3 for each treatment per each transfection. Statistical analysis was performed using a two-tailed unpaired Student’s t test. ** p < 0.01 vs. Cu/Zn-untreated medium that corresponds to the same transfection. N.S., not significant; ( d ) the extracellular levels of different folding states of hSOD1 in the mediums; ( e ) extracellular misfolded SOD1 in the medium was detected by immunoprecipitation with C4F6 followed by non-reducing Western blotting to determine the thiol/disulfide redox balance of the misfolded species. β-ME = beta-mercaptoethanol.

Article Snippet: As capture antibodies, two sets of antibodies specific for misfolded SOD1, EDI (0.5 µg/mL, SPC-206D StressMarq Biosciences, Victoria, BC, Canada) [ ] and C4F6 (×2000, MM-0070-2-P, Médimabs, Montreal, QC, Canada) [ ], were coated onto 96-well plates (Nunc-Immuno Module plate, Nunc, Roskilde, Denmark) at 4 °C for 18 h. The wells of the plates were washed three times with PBS (pH 7.4) containing 0.05% ( v / v ) Tween 20 and 5 mM EDTA, and then blocked with PBS (pH 7.4) containing 3% ( w / v ) bovine serum albumin and 5 mM EDTA at room temperature for 1 h. The NP-40 soluble faction (40 µg protein/well) or conditioned medium (40 µg protein/well) was added to the wells and incubated at room temperature for 1 h. To determine the absolute level of misfolded SOD1, metal-free hSOD1 with disulfide bonds (apo-SOD1 S-S ) was used as a standard to create a calibration curve.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: Extracellular misfolded apo-SOD1 S-S induces intracellular propagation of hSOD1 WT misfolding in recipient cells. A procedure to investigate intracellular prion-like propagation of human wild-type superoxide dismutase 1 (hSOD1 WT ) misfolding was performed using immunoprecipitation of the Nonidet P (NP-40) soluble fraction with C4F6 followed by Western blotting with antibodies to ( a , c ) SOD1 or (f) green fluorescence protein (GFP). The conditioned media from NSC-34 cells transfected with hSOD1-GFP containing misfolded apo-SOD1 S-S were used as a donor medium, whereas NSC-34 cells transfected with GFP-untagged hSOD1 WT were used as a recipient cell. ( a ) The recipient cells were exposed to conditioned medium pretreated with or without C4F6 to remove misfolded apo-SOD1 S-S ; ( b ) relative level of intracellular misfolded hSOD1 WT in the recipient cells exposed to medium pretreated with or without C4F6, ** p < 0.01 vs. C4F6-untreated cells that correspond to the same transfection, n = 3 for each treatment; ( c ) the recipient cells were pretreated with endocytosis inhibitors, 50 nM wortmannin, or 100 µM 5-( N -ethyl- N -isopropyl) amiloride (EIPA), for 1 h, and the cells were exposed to the medium for 24 h; ( d ) relative level of intracellular misfolded hSOD1 WT in endocytosis-impaired cells, ** p < 0.01 vs. DMSO-treaded cells that were exposed to the corresponding medium, n = 3 for each treatment; ( e ) the recipient cells expressing GFP-tagged hSOD1 WT were exposed to 0.5 µg/mL purified hSOD1 WT proteins with a disulfide bond (Cu/Zn SOD1 S-S , Zn SOD1 S-S , and apo-SOD1 S-S ) for 24 h, n = 3 for each treatment; ( f ) relative level of intracellular misfolded hSOD1 WT cells treated with the purified hSOD1 WT protein, ** p < 0.01 vs. HEPES-treated cells, N.S. = not significant. All data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test.

Article Snippet: As capture antibodies, two sets of antibodies specific for misfolded SOD1, EDI (0.5 µg/mL, SPC-206D StressMarq Biosciences, Victoria, BC, Canada) [ ] and C4F6 (×2000, MM-0070-2-P, Médimabs, Montreal, QC, Canada) [ ], were coated onto 96-well plates (Nunc-Immuno Module plate, Nunc, Roskilde, Denmark) at 4 °C for 18 h. The wells of the plates were washed three times with PBS (pH 7.4) containing 0.05% ( v / v ) Tween 20 and 5 mM EDTA, and then blocked with PBS (pH 7.4) containing 3% ( w / v ) bovine serum albumin and 5 mM EDTA at room temperature for 1 h. The NP-40 soluble faction (40 µg protein/well) or conditioned medium (40 µg protein/well) was added to the wells and incubated at room temperature for 1 h. To determine the absolute level of misfolded SOD1, metal-free hSOD1 with disulfide bonds (apo-SOD1 S-S ) was used as a standard to create a calibration curve.

Techniques: Immunoprecipitation, Western Blot, Fluorescence, Transfection, Expressing, Purification

Journal: International Journal of Molecular Sciences

Article Title: A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells

doi: 10.3390/ijms22084155

Figure Lengend Snippet: The conditioned medium containing extracellular misfolded metal free, disulfide oxidized superoxide dismutase 1 (apo-SOD1 S-S ) exerted cytotoxicity to motor neuron-like cells. (white) NSC-34 cells expressing green fluorescence protein (GFP) untagged human wild-type SOD1 (hSOD1 WT ) were exposed to conditioned medium from NSC-34 harboring hSOD1-GFP containing misfolded apo-SOD1 S-S for 24 h. Cell viability and cytotoxicity were assessed using ( a ) a Cell Counting Kit 8 assay and ( b ) a lactate dehydrogenase (LDH) release assay, respectively. (black) Extracellular misfolded apo-SOD1 S-S was removed from the conditioned medium by immunoprecipitation with C4F6. (gray) As a control, the medium was immunoprecipitated with normal mouse IgG. In the data set of cell viability, the results are expressed as the cell viability of recipient cells relative to that of untransfected cells, which were incubated with normal culture medium, Dulbecco’s modified Eagle’s medium and F-12 with GlutaMAX™ containing 1% ( v / v ) fetal bovine serum and 0.1 mM non-essential amino acids, instead of the conditioned medium. In the data set of cytotoxicity, the results are given as the amounts of released LDH to the medium relative to that of cells exposed to 100 μM H 2 O 2 for 24 h. All data are expressed as the mean ± SD, n = 3 for each treatment. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer post hoc test. ** p < 0.01.

Article Snippet: As capture antibodies, two sets of antibodies specific for misfolded SOD1, EDI (0.5 µg/mL, SPC-206D StressMarq Biosciences, Victoria, BC, Canada) [ ] and C4F6 (×2000, MM-0070-2-P, Médimabs, Montreal, QC, Canada) [ ], were coated onto 96-well plates (Nunc-Immuno Module plate, Nunc, Roskilde, Denmark) at 4 °C for 18 h. The wells of the plates were washed three times with PBS (pH 7.4) containing 0.05% ( v / v ) Tween 20 and 5 mM EDTA, and then blocked with PBS (pH 7.4) containing 3% ( w / v ) bovine serum albumin and 5 mM EDTA at room temperature for 1 h. The NP-40 soluble faction (40 µg protein/well) or conditioned medium (40 µg protein/well) was added to the wells and incubated at room temperature for 1 h. To determine the absolute level of misfolded SOD1, metal-free hSOD1 with disulfide bonds (apo-SOD1 S-S ) was used as a standard to create a calibration curve.

Techniques: Expressing, Fluorescence, Cell Counting, Lactate Dehydrogenase Assay, Immunoprecipitation, Incubation, Modification

Journal: Animal Models and Experimental Medicine

Article Title: Adropin modulates pancreatic cell proliferation and glutathione levels in an animal model of type 1 diabetes mellitus

doi: 10.1002/ame2.70092

Figure Lengend Snippet: Impact of adropin on superoxide dismutase expression in pancreatic β‐cells of normoglycemic and diabetic rats. (A) Immunofluorescence labeling with anti‐superoxide dismutase antibody and anti‐insulin antibodies showed expression of superoxide dismutase in pancreatic β‐cells. (B) Quantification of the histological analysis showed a significant (*** p < 0.001) decrease in superoxide dismutase distribution in pancreatic endocrine cells and a significant (**** p < 0.0001) decrease in its localization in β‐cells of rats with diabetes when compared to the normal group. n = 6. Scale bar: 50 μm. Data analysis was done using the ANOVA test.

Article Snippet: Superoxide dismutase (rabbit) (1:500) , Rockland Immunochemicals, USA.

Techniques: Expressing, Immunofluorescence, Labeling

Journal: Animal Models and Experimental Medicine

Article Title: Adropin modulates pancreatic cell proliferation and glutathione levels in an animal model of type 1 diabetes mellitus

doi: 10.1002/ame2.70092

Figure Lengend Snippet: Effect of adropin on superoxide dismutase expression in pancreatic α‐cells of normal and diabetic rats. (A) Immunofluorescence staining using anti‐superoxide dismutase and anti‐glucagon antibodies showed expression of superoxide dismutase in pancreatic α‐cells. (B) Quantification of the histological analysis showed a significant (** p < 0.01) decrease in superoxide dismutase distribution in the pancreatic endocrine cells of diabetic rats compared to the normal group. α‐Cell expression of superoxide dismutase did not change with adropin treatment among all groups. n = 6. Scale bar: 50 μm. Data analysis was done using the ANOVA test.

Article Snippet: Superoxide dismutase (rabbit) (1:500) , Rockland Immunochemicals, USA.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Animal Models and Experimental Medicine

Article Title: Adropin modulates pancreatic cell proliferation and glutathione levels in an animal model of type 1 diabetes mellitus

doi: 10.1002/ame2.70092

Figure Lengend Snippet: Effect of adropin on catalase, superoxide dismutase and total glutathione activities in the serum samples of healthy and diabetic rats. (A) Catalase activity was significantly decreased in diabetic rats compared to normal controls. Adropin slightly increased catalase in DMT group. (B) Superoxide dismutase was slightly increased in DMT compared to the diabetic treated with adropin. (C) Total glutathione was significantly raised with adropin treatment in DMT compared to DMUT. n = 4–6. Data analysis was done using the ANOVA test. * p < 0.05.

Article Snippet: Superoxide dismutase (rabbit) (1:500) , Rockland Immunochemicals, USA.

Techniques: Activity Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Aronia melanocarpa Prevents Alcohol-Induced Chronic Liver Injury via Regulation of Nrf2 Signaling in C57BL/6 Mice

doi: 10.1155/2020/4054520

Figure Lengend Snippet: Nrf2 signaling regulated by PI3K/Akt activation is involved in AM-mediated liver protection in mice with chronic alcohol injury. (a) AM and Sil increased the expression levels of P-PI3K, P-Akt, P-Stat3, Nrf2, SOD-1/2, and HO-1/2 in livers. (b) AM and Sil increased the expression levels of Nrf2 in the cytoplasm and nucleus of liver tissues. The data of quantified protein expressions were normalized by related total protein expressions, GAPDH, β -actin, and/or lamin B. The data were analyzed using one-way ANOVA and expressed as means ± S.E.M. ( n = 4). ## p < 0.01 and ### p < 0.001 vs. the control group; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 vs. the alcohol-only treated model group. AM: Aronia melanocarpa ; Sil: silybin.

Article Snippet: After blocking with 5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) at 4°C for 4 h, the membrane was incubated with primary antibody (1 : 1000 dilution) against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ABS16), total-signal transducer and activator of transcription 3 (T-Stat3; 06-596) (Merck Millipore, Burlington, Massachusetts, USA), beta-actin ( β -actin; sc-47778) (Santa Cruz, 2145 Delaware Ave, USA), phosphor- (P-) Stat3 (cstD3A7), P-phosphatidylinositol-3-hydroxykinase (PI3K; cst4228s), T-PI3K (cst4292s; Cell Signaling Technology, Boston, MA, USA), P-protein kinase B (P-Akt) (ab108266), T-Akt (ab200195), Kelch-like ECH-associated protein 1 (Keap1; ab150654), Nrf2 (ab89443), heme oxygenase-1 (HO-1; ab137749), SOD-2 (ab13533) (Abcam, Cambridge Science Park, UK), HO-2 (bs-1238R), SOD-1 (bs-10216R), and lamin B (bs-1840R) (Bioss Antibodies Biotechnology Co., Ltd., Beijing, China) overnight at 4°C.

Techniques: Activation Assay, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Differential autophagy power in the spinal cord and muscle of transgenic ALS mice

doi: 10.3389/fncel.2013.00234

Figure Lengend Snippet: Biochemical properties of wtSOD1 and mutSOD1 in spinal cord and muscle of ALS animal models . Western Blot (WB) analysis and Filter Retardation Assay (FRA) on proteins extracted from whole spinal cord ( A , WB; C , FRA) or quadriceps muscles ( B , WB; D , FRA) of female and male non-transgenic (NTg) mice, of mice expressing the wild type human SOD1 transgene (wtSOD1), and of mice expressing the G93A mutant form of human SOD1 (G93A-SOD1), at 8 (corresponding to presymptomatic stage, PS, in Tg G93A-SOD1 mice) or 16 weeks (corresponding to symptomatic stage, S, in Tg G93A-SOD1 mice). GAPDH was used to normalize protein loading. hSOD1, transgenic human SOD1; mSOD1, endogenous murine SOD1.

Article Snippet: The membranes were treated with a blocking solution containing 5% non-fat dried milk powder (EuroClone) in TBS-T for 1 h and then incubated with the primary antibodies: (1) home-made rabbit polyclonal anti-HSPB8 (kindly provided by Dr Jacques Landry, Quèbec, Canada, dilution 1:1000); (2) rabbit-polyclonal anti-BAG3 antibody (Abcam, Cambridge, UK; dilution 1:1000); (3) rabbit-polyclonal anti-BAG1 (Santa Cruz Biotechnology, Dallas, Texas, USA; dilution 1:1000); (4) rabbit polyclonal anti-LC3 (Sigma; dilution 1:1000); (5) rabbit polyclonal anti-SQSTM1/p62 (Abcam; dilution 1:1000); (6) rabbit-polyclonal anti-GAPDH (Santa Cruz Biotech; dilution 1:1000); (7) rabbit-polyclonal anti-SOD1 (SOD-100; StressMarq, Victoria, BC, Canada; dilution 1:1000).

Techniques: Western Blot, Transgenic Assay, Expressing, Mutagenesis

Journal: Frontiers in Cellular Neuroscience

Article Title: Differential autophagy power in the spinal cord and muscle of transgenic ALS mice

doi: 10.3389/fncel.2013.00234

Figure Lengend Snippet: Changes in HSPB8 levels in response to wt or mutSOD1 expression in spinal cord and muscle of ALS animal models . RT-qPCRs were performed on total RNA extracted from whole spinal cord (A) or quadriceps muscles (B) of female and male non-transgenic (NTg) mice, of mice expressing the wild type human SOD1 transgene (Tg wtSOD1), and of mice expressing the G93A mutant form of human SOD1 (Tg G93A-SOD1), at 8 (corresponding to presymptomatic stage, PS, in Tg G93A-SOD1 mice) or 16 weeks (corresponding to symptomatic stage, S, in Tg G93A-SOD1 mice). Data have been normalized to the amount of GAPDH mRNA, expressed relative to the levels determined in age-matched PS female NTg mice, taken as internal reference, and expressed as fold changes. Data are mean ± SD of four independent replicates. (A) ** p < 0.01 vs. age- and sex-matched NTg mice. (B) ** p < 0.01 vs. age- and sex-matched NTg mice and Tg wt SOD1 mice; °° p < 0.01 vs. PS (8 weeks) female Tg G93A-SOD1; # p < 0.05 vs. PS (8 weeks) male Tg G93A-SOD1.

Article Snippet: The membranes were treated with a blocking solution containing 5% non-fat dried milk powder (EuroClone) in TBS-T for 1 h and then incubated with the primary antibodies: (1) home-made rabbit polyclonal anti-HSPB8 (kindly provided by Dr Jacques Landry, Quèbec, Canada, dilution 1:1000); (2) rabbit-polyclonal anti-BAG3 antibody (Abcam, Cambridge, UK; dilution 1:1000); (3) rabbit-polyclonal anti-BAG1 (Santa Cruz Biotechnology, Dallas, Texas, USA; dilution 1:1000); (4) rabbit polyclonal anti-LC3 (Sigma; dilution 1:1000); (5) rabbit polyclonal anti-SQSTM1/p62 (Abcam; dilution 1:1000); (6) rabbit-polyclonal anti-GAPDH (Santa Cruz Biotech; dilution 1:1000); (7) rabbit-polyclonal anti-SOD1 (SOD-100; StressMarq, Victoria, BC, Canada; dilution 1:1000).

Techniques: Expressing, Transgenic Assay, Mutagenesis, Mouse Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Differential autophagy power in the spinal cord and muscle of transgenic ALS mice

doi: 10.3389/fncel.2013.00234

Figure Lengend Snippet: BAG3 and BAG1 expression in spinal cord and muscle of ALS animal models . RT-qPCRs were performed on total RNA extracted from whole spinal cord (A,C) or quadriceps muscles (B,D) of female and male non-transgenic (NTg) mice, of mice expressing the wild type human SOD1 transgene (Tg wtSOD1), and of mice expressing the G93A mutant form of human SOD1 (Tg G93A-SOD1), at 8 (corresponding to presymptomatic stage, PS, in Tg G93A-SOD1 mice) or 16 weeks (corresponding to symptomatic stage, S, in Tg G93A-SOD1 mice). Data have been normalized to the amount of GAPDH mRNA, expressed relative to the levels determined in age-matched PS female NTg mice, taken as internal reference, and expressed as fold changes. Data are mean ± SD of four independent replicates. (A) RT-qPCR on BAG3 mRNA expression levels in whole spinal cord. ** p < 0.01 vs. age- and sex-matched NTg and Tg wtSOD1 mice; ∧ p < 0.05 vs. age- and sex-matched NTg mice; §§ p < 0.01 vs. age- and sex-matched Tg wtSOD1 mice. (B) RT-qPCR on BAG3 mRNA expression levels in quadriceps muscles. ** p < 0.01 vs. age- and sex-matched NTg and Tg wtSOD1 mice; ° p < 0.05 vs. PS (8 weeks) female Tg G93A-SOD1; ## p < 0.01 vs. PS (8 weeks) male Tg G93A-SOD1. (C) RT-qPCR on BAG1 mRNA expression levels in whole spinal cord. (D) RT-qPCR on BAG1 mRNA expression levels in quadriceps muscles. ** p < 0.01 vs. age- and sex-matched NTg and Tg wtSOD1 mice; ° p < 0.05 vs. sex-matched PS (8 weeks) Tg G93A-SOD1. (E) BAG3:BAG1 relative ratio of mRNA expression levels in whole spinal cord. Data represent variations of the relative levels of BAG3 and BAG1 normalized over the relative BAG3 and BAG1 levels of age-matched PS (8 weeks) female NTg mice (taken as internal reference, see Materials and Methods for details). ** p < 0.01 vs. age- and sex-matched NTg and Tg wtSOD1 mice; ## p < 0.01 vs. PS (8 weeks) male Tg G93A-SOD1. (F) BAG3:BAG1 relative ratio of mRNA expression levels in quadriceps muscles. Data have been calculated as in (E) . * p < 0.05 and ** p < 0.01 vs. age- and sex-matched NTg and Tg wtSOD1 mice; ° p < 0.05 vs. PS (8 weeks) male Tg G93A-SOD1.

Article Snippet: The membranes were treated with a blocking solution containing 5% non-fat dried milk powder (EuroClone) in TBS-T for 1 h and then incubated with the primary antibodies: (1) home-made rabbit polyclonal anti-HSPB8 (kindly provided by Dr Jacques Landry, Quèbec, Canada, dilution 1:1000); (2) rabbit-polyclonal anti-BAG3 antibody (Abcam, Cambridge, UK; dilution 1:1000); (3) rabbit-polyclonal anti-BAG1 (Santa Cruz Biotechnology, Dallas, Texas, USA; dilution 1:1000); (4) rabbit polyclonal anti-LC3 (Sigma; dilution 1:1000); (5) rabbit polyclonal anti-SQSTM1/p62 (Abcam; dilution 1:1000); (6) rabbit-polyclonal anti-GAPDH (Santa Cruz Biotech; dilution 1:1000); (7) rabbit-polyclonal anti-SOD1 (SOD-100; StressMarq, Victoria, BC, Canada; dilution 1:1000).

Techniques: Expressing, Transgenic Assay, Mutagenesis, Quantitative RT-PCR, Mouse Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Differential autophagy power in the spinal cord and muscle of transgenic ALS mice

doi: 10.3389/fncel.2013.00234

Figure Lengend Snippet: HSPB8-BAG1-BAG3 protein levels in spinal cord and muscle of ALS animal models . Western Blot (WB) analysis on proteins extracted from whole spinal cord (A) or quadriceps muscles (B) of female and male non-transgenic (NTg) mice, of mice expressing the wild type human SOD1 transgene (Tg wtSOD1), and of mice expressing the G93A mutant form of human SOD1 (Tg G93A-SOD1), at 8 (corresponding to presymptomatic stage, PS, in Tg G93A-SOD1 mice) or 16 weeks (corresponding to symptomatic stage, S, in Tg G93A-SOD1 mice). GAPDH was used to normalize protein loading.

Article Snippet: The membranes were treated with a blocking solution containing 5% non-fat dried milk powder (EuroClone) in TBS-T for 1 h and then incubated with the primary antibodies: (1) home-made rabbit polyclonal anti-HSPB8 (kindly provided by Dr Jacques Landry, Quèbec, Canada, dilution 1:1000); (2) rabbit-polyclonal anti-BAG3 antibody (Abcam, Cambridge, UK; dilution 1:1000); (3) rabbit-polyclonal anti-BAG1 (Santa Cruz Biotechnology, Dallas, Texas, USA; dilution 1:1000); (4) rabbit polyclonal anti-LC3 (Sigma; dilution 1:1000); (5) rabbit polyclonal anti-SQSTM1/p62 (Abcam; dilution 1:1000); (6) rabbit-polyclonal anti-GAPDH (Santa Cruz Biotech; dilution 1:1000); (7) rabbit-polyclonal anti-SOD1 (SOD-100; StressMarq, Victoria, BC, Canada; dilution 1:1000).

Techniques: Western Blot, Transgenic Assay, Expressing, Mutagenesis

Journal: Frontiers in Cellular Neuroscience

Article Title: Differential autophagy power in the spinal cord and muscle of transgenic ALS mice

doi: 10.3389/fncel.2013.00234

Figure Lengend Snippet: LC3 and p62 expression in spinal cord and muscle of ALS animal models . RT-qPCRs were performed on total RNA extracted from whole spinal cord (A,C) or quadriceps muscles (B,D) of female and male non-transgenic (NTg) mice, of mice expressing the wild type human SOD1 transgene (Tg wtSOD1), and of mice expressing the G93A mutant form of human SOD1 (Tg G93A-SOD1), at 8 (corresponding to presymptomatic stage, PS, in Tg G93A-SOD1 mice) or 16 weeks (corresponding to symptomatic stage, S, in Tg G93A-SOD1 mice). Data have been normalized to the amount of GAPDH mRNA, expressed relative to the levels determined in age-matched PS female NTg mice, taken as internal reference, and expressed as fold changes. Data are mean ± SD of four independent replicates. (A) RT-qPCR on LC3 mRNA expression levels in whole spinal cord. ** p < 0.01 vs. age- and sex-matched NTg and Tg wtSOD1 mice. (B) RT-qPCR on LC3 mRNA expression levels in quadriceps muscles. ∧ p < 0.05 vs. age- and sex-matched NTg mice; §§ p < 0.01 vs. age- and sex-matched Tg wtSOD1 mice. (C) RT-qPCR on p62 mRNA expression levels in whole spinal cord. (D) RT-qPCR on p62 mRNA expression levels in quadriceps muscles. ** p < 0.01 vs. age- and sex-matched NTg and Tg wtSOD1 mice; °° p < 0.01 vs. PS female (8 weeks) Tg G93A-SOD1.

Article Snippet: The membranes were treated with a blocking solution containing 5% non-fat dried milk powder (EuroClone) in TBS-T for 1 h and then incubated with the primary antibodies: (1) home-made rabbit polyclonal anti-HSPB8 (kindly provided by Dr Jacques Landry, Quèbec, Canada, dilution 1:1000); (2) rabbit-polyclonal anti-BAG3 antibody (Abcam, Cambridge, UK; dilution 1:1000); (3) rabbit-polyclonal anti-BAG1 (Santa Cruz Biotechnology, Dallas, Texas, USA; dilution 1:1000); (4) rabbit polyclonal anti-LC3 (Sigma; dilution 1:1000); (5) rabbit polyclonal anti-SQSTM1/p62 (Abcam; dilution 1:1000); (6) rabbit-polyclonal anti-GAPDH (Santa Cruz Biotech; dilution 1:1000); (7) rabbit-polyclonal anti-SOD1 (SOD-100; StressMarq, Victoria, BC, Canada; dilution 1:1000).

Techniques: Expressing, Transgenic Assay, Mutagenesis, Quantitative RT-PCR, Mouse Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Differential autophagy power in the spinal cord and muscle of transgenic ALS mice

doi: 10.3389/fncel.2013.00234

Figure Lengend Snippet: Autophagic-markers (p62 and LC-3) protein levels in spinal cord and muscle of ALS animal models . Western Blot (WB) analysis on proteins extracted from whole spinal cord (A) or quadriceps muscles (B) of female and male non-transgenic (NTg) mice, of mice expressing the wild type human SOD1 transgene (Tg wtSOD1), and of mice expressing the G93A mutant form of human SOD1 (Tg G93A-SOD1), at 8 (corresponding to presymptomatic stage, PS, in Tg G93A-SOD1 mice) or 16 weeks (corresponding to symptomatic stage, S, in Tg G93A-SOD1 mice). GAPDH was used to normalize protein loading.

Article Snippet: The membranes were treated with a blocking solution containing 5% non-fat dried milk powder (EuroClone) in TBS-T for 1 h and then incubated with the primary antibodies: (1) home-made rabbit polyclonal anti-HSPB8 (kindly provided by Dr Jacques Landry, Quèbec, Canada, dilution 1:1000); (2) rabbit-polyclonal anti-BAG3 antibody (Abcam, Cambridge, UK; dilution 1:1000); (3) rabbit-polyclonal anti-BAG1 (Santa Cruz Biotechnology, Dallas, Texas, USA; dilution 1:1000); (4) rabbit polyclonal anti-LC3 (Sigma; dilution 1:1000); (5) rabbit polyclonal anti-SQSTM1/p62 (Abcam; dilution 1:1000); (6) rabbit-polyclonal anti-GAPDH (Santa Cruz Biotech; dilution 1:1000); (7) rabbit-polyclonal anti-SOD1 (SOD-100; StressMarq, Victoria, BC, Canada; dilution 1:1000).

Techniques: Western Blot, Transgenic Assay, Expressing, Mutagenesis

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