Journal: BioMed Research International

Article Title: Accumulation of Misfolded SOD1 in Dorsal Root Ganglion Degenerating Proprioceptive Sensory Neurons of Transgenic Mice with Amyotrophic Lateral Sclerosis

doi: 10.1155/2014/852163

Figure Lengend Snippet: Morphometric analysis of motor (L4 VRs, (a)–(f)) and sensory (L4 DRs, (g)–(l)) axons from WT and SOD1 G93A mice. An analysis was performed on 1 μ m semithin plastic transversal sections ((b), (c), (h), and (i)). ((a)–(c) and (g)–(i)) Counts showed a significant loss of the total number of motor, but not sensory, axons in the end-stage (P120) SOD1 G93A mice ((a) and (g)). ((d)–(f) and (j)–(l)) A frequency distribution analysis of myelinated axon size in WT VRs showed a clear bimodal profile (f) indicative of axons coming from α -MNs (large) and γ -MNs (small); note the selective loss of large axons in SOD1 G93A mice. Representative images of WT and SOD1 G93A ventral nerve are shown in (d) and (e); note the presence of abundant degenerating axons in SOD1 G93A animals. Although there was no evidence of the loss of sensory axons in dorsal nerve roots in SOD1 G93A mice, a more detailed examination of nerve profile morphology ((j) and (k)) revealed the presence of moderate numbers of axons exhibiting significant degrees of swelling (∗) and other degenerating features. A frequency distribution analysis of myelinated axon size in dorsal nerve roots (l) reflected the appearance of a new population of large diameter (degenerating) sensory axons in SOD1 G93A samples (indicated by a double arrowed line). The bars in the graphs represent the mean ± SEM values of counts performed in 2–11 animals per age and experimental condition; ** P < 0.01 versus WT, one-way ANOVA, Bonferroni's post hoc test). Scale bar in D = 10 μ m (valid for (e), (j), and (k)).

Article Snippet: The primary antibodies used were the following: (1) AJ10 antibody was produced in our laboratory by immunizing rabbits with a human SOD1 peptide sequence (VKVWGSIKGLTEGLHGFHVHEFGDNTAGC); this antibody is specific for misfolded ALS-linked mutant human SOD1conformers and barely reacts with wild-type (WT) human SOD1 [ ] rabbit polyclonal anti-P2X4 (1 : 500; Alomone Labs, Jerusalem, Israel); some lots of this antibody, such as lot AN-04 and AN-05, cross-react with high affinity with ALS-linked misfolded conformers of SOD1 [ , ]; (3) rat monoclonal anti-Mac-2 (1 : 800, Antibodies online GMBH, Aachen, Germany); (4) rat monoclonal anti-mouse CD68 (1 : 100; AbD Serotec, Oxford, UK); (5) mouse monoclonal anti-calcitonin gene-related peptide (CGRP, 1 : 100; Abcam, Cambridge, UK; ab81887); (6) goat polyclonal anti-parvalbumin (PV, 1 : 1000; Swant, Marly, Switzerland; pv-235); (7) guinea pig polyclonal anti-substance P (SP, 1 : 1000; Abcam; ab10353); and (8) mouse monoclonal anti-neuron-specific class III β -tubulin (TUJ1, 1 : 500, RD Systems, Minneapolis, MN, USA; mab1195).

Techniques:

Journal: BioMed Research International

Article Title: Accumulation of Misfolded SOD1 in Dorsal Root Ganglion Degenerating Proprioceptive Sensory Neurons of Transgenic Mice with Amyotrophic Lateral Sclerosis

doi: 10.1155/2014/852163

Figure Lengend Snippet: The ultrastructural morphology of VRs ((a), (c), and (d)) and DRs (b) from end-stage SOD1 G93A mice. From a qualitative point of view, similar Wallerian-like degenerative changes could be observed in both VRs (a) and DRs (b); phagocytic cells (shaded in green), in which the nucleus is dashed in blue, appear to engulf the myelin debris (shaded in red); apparently normal myelinated axons are dashed in yellow and Schwann cell profiles are dashed in magenta. ((c)–(d)) In ventral roots it is possible to observe some regenerating axons (yellow in (c)) growing inside the ghosts of folded basal lamina profiles (green in (c)) filled with Schwann cell pedicles (magenta in (c)). Occasionally, a large growth cone (yellow in (d)) filled with organelles and surrounded by Schwann cell profiles (magenta in (d)) can be detected. Scale bars: 5 μ m in (a), 2.5 μ m in (b), 0.5 μ m in (c), and 1 μ m in (d).

Article Snippet: The primary antibodies used were the following: (1) AJ10 antibody was produced in our laboratory by immunizing rabbits with a human SOD1 peptide sequence (VKVWGSIKGLTEGLHGFHVHEFGDNTAGC); this antibody is specific for misfolded ALS-linked mutant human SOD1conformers and barely reacts with wild-type (WT) human SOD1 [ ] rabbit polyclonal anti-P2X4 (1 : 500; Alomone Labs, Jerusalem, Israel); some lots of this antibody, such as lot AN-04 and AN-05, cross-react with high affinity with ALS-linked misfolded conformers of SOD1 [ , ]; (3) rat monoclonal anti-Mac-2 (1 : 800, Antibodies online GMBH, Aachen, Germany); (4) rat monoclonal anti-mouse CD68 (1 : 100; AbD Serotec, Oxford, UK); (5) mouse monoclonal anti-calcitonin gene-related peptide (CGRP, 1 : 100; Abcam, Cambridge, UK; ab81887); (6) goat polyclonal anti-parvalbumin (PV, 1 : 1000; Swant, Marly, Switzerland; pv-235); (7) guinea pig polyclonal anti-substance P (SP, 1 : 1000; Abcam; ab10353); and (8) mouse monoclonal anti-neuron-specific class III β -tubulin (TUJ1, 1 : 500, RD Systems, Minneapolis, MN, USA; mab1195).

Techniques:

Journal: BioMed Research International

Article Title: Accumulation of Misfolded SOD1 in Dorsal Root Ganglion Degenerating Proprioceptive Sensory Neurons of Transgenic Mice with Amyotrophic Lateral Sclerosis

doi: 10.1155/2014/852163

Figure Lengend Snippet: ((a)–(c)) Misfolded SOD1 (misSOD1) immunolabeling using AJ10 antibody (red) reveals highly immunoreactive neuronal somata within the DRG (delimited in (a)) of end-stage (P120) SOD1 G93A mice. Some neuronal somata display cytoplasmic fragmentation indicative of degenerative changes (arrows in (b)); nuclei, counterstained with DAPI (blue), do not show apoptotic morphology (arrows in (c)). ((d)–(g)) Misfolded SOD1 immunolabeling (red) was combined with an anti-CD68 antibody (green) to reveal activated macrophagic cells; DAPI (blue) was used for nuclear staining. A degenerating neuron expressing misfolded SOD1 (delimited in (d)) and displaying clustered profiles of CD68 positive phagocytic cells is shown in high magnification in (e)–(g). (h) A frequency distribution profile of the size of the DRG neuron somata containing misfolded SOD1 with respect to that of the whole neuronal population; note that misfolded SOD1 positive neurons belong to the large-sized (presumably proprioceptive) population. Scale bars: 80 μ m in (a), 40 μ m in (b), (c), (d), and (g) (also valid for (e) and (f)).

Article Snippet: The primary antibodies used were the following: (1) AJ10 antibody was produced in our laboratory by immunizing rabbits with a human SOD1 peptide sequence (VKVWGSIKGLTEGLHGFHVHEFGDNTAGC); this antibody is specific for misfolded ALS-linked mutant human SOD1conformers and barely reacts with wild-type (WT) human SOD1 [ ] rabbit polyclonal anti-P2X4 (1 : 500; Alomone Labs, Jerusalem, Israel); some lots of this antibody, such as lot AN-04 and AN-05, cross-react with high affinity with ALS-linked misfolded conformers of SOD1 [ , ]; (3) rat monoclonal anti-Mac-2 (1 : 800, Antibodies online GMBH, Aachen, Germany); (4) rat monoclonal anti-mouse CD68 (1 : 100; AbD Serotec, Oxford, UK); (5) mouse monoclonal anti-calcitonin gene-related peptide (CGRP, 1 : 100; Abcam, Cambridge, UK; ab81887); (6) goat polyclonal anti-parvalbumin (PV, 1 : 1000; Swant, Marly, Switzerland; pv-235); (7) guinea pig polyclonal anti-substance P (SP, 1 : 1000; Abcam; ab10353); and (8) mouse monoclonal anti-neuron-specific class III β -tubulin (TUJ1, 1 : 500, RD Systems, Minneapolis, MN, USA; mab1195).

Techniques: Immunolabeling, Staining, Expressing

Journal: BioMed Research International

Article Title: Accumulation of Misfolded SOD1 in Dorsal Root Ganglion Degenerating Proprioceptive Sensory Neurons of Transgenic Mice with Amyotrophic Lateral Sclerosis

doi: 10.1155/2014/852163

Figure Lengend Snippet: Micrographs taken from the DRGs of end-stage (P120) SOD1 G93A mice. Misfolded SOD1 (misSOD1) immunolabeling using AJ10 antibody (red) was combined with several markers for DRG neuronal types, as indicated (green). ((a)–(i)) Misfolded SOD1 immunoreactive neurons do not colocalize with the neuropeptides CGRP and SP, or with IB4, all of which are markers for neuronal types other than proprioceptive. ((j)–(l)) However, neurons containing misfolded SOD1 colocalize with PV, a marker for proprioceptive sensory neurons. In some cases, DAPI was used for nuclear staining (blue). The prominent background observed in (c) and (d) is due to an unspecific connective tissue reaction involving the secondary antibody when a mouse monoclonal was used as a primary antibody. Scale bars = 200 μ m.

Article Snippet: The primary antibodies used were the following: (1) AJ10 antibody was produced in our laboratory by immunizing rabbits with a human SOD1 peptide sequence (VKVWGSIKGLTEGLHGFHVHEFGDNTAGC); this antibody is specific for misfolded ALS-linked mutant human SOD1conformers and barely reacts with wild-type (WT) human SOD1 [ ] rabbit polyclonal anti-P2X4 (1 : 500; Alomone Labs, Jerusalem, Israel); some lots of this antibody, such as lot AN-04 and AN-05, cross-react with high affinity with ALS-linked misfolded conformers of SOD1 [ , ]; (3) rat monoclonal anti-Mac-2 (1 : 800, Antibodies online GMBH, Aachen, Germany); (4) rat monoclonal anti-mouse CD68 (1 : 100; AbD Serotec, Oxford, UK); (5) mouse monoclonal anti-calcitonin gene-related peptide (CGRP, 1 : 100; Abcam, Cambridge, UK; ab81887); (6) goat polyclonal anti-parvalbumin (PV, 1 : 1000; Swant, Marly, Switzerland; pv-235); (7) guinea pig polyclonal anti-substance P (SP, 1 : 1000; Abcam; ab10353); and (8) mouse monoclonal anti-neuron-specific class III β -tubulin (TUJ1, 1 : 500, RD Systems, Minneapolis, MN, USA; mab1195).

Techniques: Immunolabeling, Marker, Staining

Journal: BioMed Research International

Article Title: Accumulation of Misfolded SOD1 in Dorsal Root Ganglion Degenerating Proprioceptive Sensory Neurons of Transgenic Mice with Amyotrophic Lateral Sclerosis

doi: 10.1155/2014/852163

Figure Lengend Snippet: (a) Semithin plastic section of a DRG from end-stage (P120) SOD1 G93A mice showing a degenerating, microvacuolated, large neuronal soma, which is surrounded by apparently normal medium-sized neuronal cells. ((b) and (c)) The cells indicated in the semithin sections were examined by electron microscopy which revealed a well-preserved organelle structure in the medium-sized neuronal cell (b) and extensive microvacuolization in the degenerating large soma (c). (d) A large myelinated axonal profile adjacent to the DRG sensory neurons shows an accumulation of highly vacuolated mitochondria with a comparable morphology to that of degenerating mitochondria typically described in motor axons of SOD1 G93A mice (as shown in (e), taken from a sample of facial nucleus). Scale bars: 20 μ m in (a), 0.5 μ m in (b) and (c), and 1.5 μ m in (d) and (e).

Article Snippet: The primary antibodies used were the following: (1) AJ10 antibody was produced in our laboratory by immunizing rabbits with a human SOD1 peptide sequence (VKVWGSIKGLTEGLHGFHVHEFGDNTAGC); this antibody is specific for misfolded ALS-linked mutant human SOD1conformers and barely reacts with wild-type (WT) human SOD1 [ ] rabbit polyclonal anti-P2X4 (1 : 500; Alomone Labs, Jerusalem, Israel); some lots of this antibody, such as lot AN-04 and AN-05, cross-react with high affinity with ALS-linked misfolded conformers of SOD1 [ , ]; (3) rat monoclonal anti-Mac-2 (1 : 800, Antibodies online GMBH, Aachen, Germany); (4) rat monoclonal anti-mouse CD68 (1 : 100; AbD Serotec, Oxford, UK); (5) mouse monoclonal anti-calcitonin gene-related peptide (CGRP, 1 : 100; Abcam, Cambridge, UK; ab81887); (6) goat polyclonal anti-parvalbumin (PV, 1 : 1000; Swant, Marly, Switzerland; pv-235); (7) guinea pig polyclonal anti-substance P (SP, 1 : 1000; Abcam; ab10353); and (8) mouse monoclonal anti-neuron-specific class III β -tubulin (TUJ1, 1 : 500, RD Systems, Minneapolis, MN, USA; mab1195).

Techniques: Electron Microscopy

Journal: BioMed Research International

Article Title: Accumulation of Misfolded SOD1 in Dorsal Root Ganglion Degenerating Proprioceptive Sensory Neurons of Transgenic Mice with Amyotrophic Lateral Sclerosis

doi: 10.1155/2014/852163

Figure Lengend Snippet: ((a)–(d)) Double immunolabeling with TUJ1 (to label neuronal profiles, red), and anti-CD68 (to reveal microglial cells, green) antibodies in ventral ((a), (c)) and dorsal ((b), (d)) horn of WT ((a), (b)) and end-stage SOD1G93A ((c), (d)) mice; L: lateral, M: medial. CD68-positive profiles were rarely detectable in WT samples, but they were present in either large or moderate quantities in the ventral and dorsal horn, respectively, of SOD1 G93A samples. In SOD1 G93A dorsal horns, it was possible to observe some infiltrating microglial cells adjacent to medially located (presumably proprioceptive) axon fascicles, entering the spinal cord (arrows in (d)). (e) Quantification of CD68-positive profiles in the spinal cord of WT and end-stage SOD1 G93A mice. Bars represent mean ± SEM values for counts performed in 6–11 fields of 2 animals per experimental condition; *** P < 0.001 versus WT (one-way ANOVA, Bonferroni's post hoc test). Scale bar in (b) = 100 μ m (also valid for (a), (c), and (d)).

Article Snippet: The primary antibodies used were the following: (1) AJ10 antibody was produced in our laboratory by immunizing rabbits with a human SOD1 peptide sequence (VKVWGSIKGLTEGLHGFHVHEFGDNTAGC); this antibody is specific for misfolded ALS-linked mutant human SOD1conformers and barely reacts with wild-type (WT) human SOD1 [ ] rabbit polyclonal anti-P2X4 (1 : 500; Alomone Labs, Jerusalem, Israel); some lots of this antibody, such as lot AN-04 and AN-05, cross-react with high affinity with ALS-linked misfolded conformers of SOD1 [ , ]; (3) rat monoclonal anti-Mac-2 (1 : 800, Antibodies online GMBH, Aachen, Germany); (4) rat monoclonal anti-mouse CD68 (1 : 100; AbD Serotec, Oxford, UK); (5) mouse monoclonal anti-calcitonin gene-related peptide (CGRP, 1 : 100; Abcam, Cambridge, UK; ab81887); (6) goat polyclonal anti-parvalbumin (PV, 1 : 1000; Swant, Marly, Switzerland; pv-235); (7) guinea pig polyclonal anti-substance P (SP, 1 : 1000; Abcam; ab10353); and (8) mouse monoclonal anti-neuron-specific class III β -tubulin (TUJ1, 1 : 500, RD Systems, Minneapolis, MN, USA; mab1195).

Techniques: Immunolabeling

Journal: Frontiers in Molecular Biosciences

Article Title: Accumulated ROS Activates HIF-1α-Induced Glycolysis and Exerts a Protective Effect on Sensory Hair Cells Against Noise-Induced Damage

doi: 10.3389/fmolb.2021.806650

Figure Lengend Snippet: Gene-specific primers in this study.

Article Snippet: The following antibodies were used: anti-SOD1 (rabbit monoclonal, ab51254, Abcam), anti-SOD2 (rabbit monoclonal, ab68155, Abcam), anti-Catalase (rabbit polyclonal, 21260-1-AP, Proteintech), anti-GPx1 (rabbit monoclonal, ab108429, Abcam), anti-GLUT1 (mouse monoclonal, ab238050, Abcam), anti-HK2 (rabbit monoclonal, ab209847, Abcam), anti-ENO1 (rabbit monoclonal, ab227978, Abcam), anti-PDK1 (rabbit monoclonal, ab202468, Abcam), anti-LDHA (mouse monoclonal, 66287-1-Ig, Proteintech), anti-HIF-1α (rabbit monoclonal, ab179483, Abcam; mouse monoclonal, 66730-1-Ig, Proteintech), anti-β-Tubulin (rabbit polyclonal, 10068-1-AP, Proteintech), anti-Myosin VIIa (rabbit monoclonal, ab155984, Abcam), and 4-HNE (mouse monoclonal, ab48506, Abcam).

Techniques:

Journal: Biology

Article Title: Neuroprotective, Antioxidant and Anti-Inflammatory Effect of Greek Pomegranate Seed Oil on N2a Neuroblastoma Cells and Mild Cognitive Impairment Patients

doi: 10.3390/biology14050548

Figure Lengend Snippet: ( a ) Representative Western blot images showed the expression levels of superoxide dismutase 1 (SOD1) across different experimental conditions (see ). ( b ) Quantification of SOD1 protein levels normalized to β-actin. ( c ) Measurement of malondialdehyde (MDA) levels under oxidative stress conditions induced by 100 µM H 2 O 2 . Untreated and H 2 O 2 -treated groups were analyzed in the presence of different PSO concentrations (0, 0.2, 10, and 25 µg/mL). Statistical significance: * p < 0.05; *** p < 0.001, with asterisk (*) indicating the comparison versus control group; ## p < 0.01, ### p < 0.001, #### p < 0.0001, with (#) indicating the comparison versus H 2 O 2 ; $ p < 0.05, with ($) indicating the comparison versus 0 µg/mL PSO. Data are presented as the mean ± SD.

Article Snippet: For the detection of APP, Aβ 42 , tau, p-tau181, iNOS, IL-1β, TNF-α and SOD1, the following primary antibodies were employed: anti-APP mouse monoclonal antibody (#60342-1-Ig, Proteintech, Manchester, UK), anti-Aβ 42 rabbit monoclonal antibody (#14974, Cell Signaling Technology, Danvers, MA, USA), anti-rabbit tau monoclonal antibody (#46687, Cell Signaling Technology, Danvers, MA, USA), anti-p-tau181 rabbit monoclonal antibody (#12885, Cell Signaling Technology, Danvers, MA, USA), anti-iNOS rabbit polyclonal antibody (#18985-1-AP, Proteintech, Manchester, UK), anti-IL-1β rabbit monoclonal antibody (#L0328Y, Cusabio, Houston, TX, USA), anti-TNF-α mouse monoclonal antibody (#sc-52746, Santa Cruz Biotechnology, Heidelberg, Germany), and an anti-SOD1 rabbit polyclonal antibody (#10269-1-AP, Proteintech, Manchester, UK).

Techniques: Western Blot, Expressing, Comparison, Control

Journal: Stem Cells Translational Medicine

Article Title: Dysregulation of Mesenchymal Stromal Cell Antioxidant Responses in Progressive Multiple Sclerosis

doi: 10.1002/sctm.18-0045

Figure Lengend Snippet: Reduced MS‐MSC secretion of SOD1 and GSTP1 in association with duration of progressive phase of MS. Analysis of the MSC secretome was undertaken using LC‐MSMS (C‐MSC n = 4; MS‐MSC n = 6) and ELISA (C‐MSC n = 5; MS‐MSC n = 11). (A): When the difference in age between the cohorts was accounted for, an independent, statistically significant effect of the presence of MS was seen with reduced secretion of SOD1 (regression analysis, #, p = .042, CI −0.093 to 3.9) as measured by LC‐MSMS. (B): Reduced secretion of SOD1 by MS‐MSC as determined by ELISA (regression analysis, ###, p < .001, CI −1.554 to −0.451). (C): SOD1 secretion by MS‐MSC as determined by ELISA negatively correlated with duration of the progressive phase of MS (Pearson r −.789, p = .002, CI −0.938 to −0.392). (D): Reduction in secretion of GSTP1 by MS‐MSC as determined by LC‐MSMS (regression analysis, #, p = .034, CI −0.975 to −0.039). (E): Reduction in GSTP1 in MS‐MSC measured by ELISA (regression analysis, #, p = .04, CI −45.332 to −1.021). (F): Negative correlation of GSTP1 secretion by MS‐MSC with duration of progressive disease (Pearson r −.665, p = .036, CI −0.913 to −0.061). The data are the means ± SEM of multiple biological replicates as listed. Abbreviations: CI, confidence interval; C‐MSC, control mesenchymal stromal cells; ELISA, enzyme‐linked immunosorbent assay; GSTP1, Glutathione S‐Transferase Pi 1; LC‐MSMS, liquid chromatography‐tandem mass spectrometry; MS, multiple sclerosis; MSC, mesenchymal stromal cells; MS‐MSC, mesenchymal stromal cells from patients with multiple sclerosis; SOD1, superoxide dismutase 1.

Article Snippet: Antibodies used were mouse anti‐SOD1 (1:2,000, R&D USA), mouse anti‐GSTP1 (1:2,000; Santa Cruz USA), rabbit anti‐peroxisome proliferator‐activated receptor‐gamma coactivator‐1alpha (PGC1α) (1:3,000; Santa Cruz), rabbit anti‐nuclear factor erythroid 2‐related factor 2 (Nrf2) (1:3,000; Santa Cruz), rabbit anticatalase (1:5,000; Abcam UK), mouse anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (1:5,000; Abcam), and antiactin (1:5,000; Abcam).

Techniques: Enzyme-linked Immunosorbent Assay, Liquid Chromatography, Mass Spectrometry

Journal: Stem Cells Translational Medicine

Article Title: Dysregulation of Mesenchymal Stromal Cell Antioxidant Responses in Progressive Multiple Sclerosis

doi: 10.1002/sctm.18-0045

Figure Lengend Snippet: MSC expression and activity of antioxidants in standard culture conditions. Densitometric analysis of Western blots was used to examine protein expression in standard culture conditions (C‐MSC n = 3; MS‐MSC n = 7). (A): SOD1 expression was reduced in MS‐MSC compared to C‐MSC (regression analysis, ##, p = .002, CI −0.737 to −0.159). (B): GSTP1 expression was reduced in MS‐MSC (regression analysis, ##, p = .006, CI −1.39 to −0.23). (C): Catalase expression was unaltered. (D): Representative bands are shown. (E): SOD activity was reduced in MS‐MSC ( n = 4) compared to C‐MSC ( n = 4; regression analysis, ##, p = .003, CI −25.706 to −5.288). (F): GST activity was reduced in MS‐MSC ( n = 6) compared to C‐MSC ( n = 3; Mann Whitney test, *, p = .038; regression analysis, ##, p = .006, CI −0.854 to −0.14). The data are the means ± SEM of multiple biological replicates as listed. Abbreviations: CI, confidence interval; C‐MSC, control mesenchymal stromal cells; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GSTP1, Glutathione S‐Transferase Pi 1; MS, multiple sclerosis; MSC, mesenchymal stromal cell; MS‐MSC, mesenchymal stromal cells from patients with multiple sclerosis; SOD1, superoxide dismutase 1.

Article Snippet: Antibodies used were mouse anti‐SOD1 (1:2,000, R&D USA), mouse anti‐GSTP1 (1:2,000; Santa Cruz USA), rabbit anti‐peroxisome proliferator‐activated receptor‐gamma coactivator‐1alpha (PGC1α) (1:3,000; Santa Cruz), rabbit anti‐nuclear factor erythroid 2‐related factor 2 (Nrf2) (1:3,000; Santa Cruz), rabbit anticatalase (1:5,000; Abcam UK), mouse anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (1:5,000; Abcam), and antiactin (1:5,000; Abcam).

Techniques: Expressing, Activity Assay, Western Blot, MANN-WHITNEY

Journal: Stem Cells Translational Medicine

Article Title: Dysregulation of Mesenchymal Stromal Cell Antioxidant Responses in Progressive Multiple Sclerosis

doi: 10.1002/sctm.18-0045

Figure Lengend Snippet: Reduced MS‐MSC expression of SOD1 and GSTP1 under conditions of nitrosative stress. Following exposure to DETANONOate, SOD1 and GSTP1 gene and protein expression relative to baseline were examined over a 24‐hour period. (A): SOD1 expression, measured by RT‐PCR relative to GAPDH, increased over time with exposure to DETANONOate in both C‐MSC and MS‐MSC (regression analysis, ##, p = .008, CI 0.002 to 0.016). There was no difference between SOD1 expression in C‐MSC and MS‐MSC at any time point examined. (B): Protein expression of SOD1 in response to nitrostative stress in C‐MSC and MS‐MSC was examined using immunoblotting. Following adjustment for age, SOD1 expression declined with exposure to DETANONOate (regression analysis, ###, p < .001, CI −0.027 to −0.019). An independent, negative effect on SOD1 expression attributable to progressive MS was seen following adjustment for age (regression analysis, p < .001, CI −0.515 to −0.226). (C): A significant change in GSTP1 expression was not observed in either C‐MSC or MS‐MSC following exposure to DETANONOate including after correction for age. (D): Overall, GSTP1 protein expression following exposure to DETANONOate fell in both C‐MSC and MS‐MSC and an independent negative effect of progressive MS was observed (regression analysis, ###, p < .001, CI −0.47 to −0.326). The data are the means ± SEM of multiple biological replicates C‐MSC n = 3, MS‐MSC n = 4. Abbreviations: CI, confidence interval; C‐MSC, control mesenchymal stromal cells; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GSTP1, Glutathione S‐Transferase Pi 1; MS, multiple sclerosis; MS‐MSC, mesenchymal stromal cells from patients with multiple sclerosis; RT‐PCR, real time polymerase chain reaction; SOD1, superoxide dismutase 1.

Article Snippet: Antibodies used were mouse anti‐SOD1 (1:2,000, R&D USA), mouse anti‐GSTP1 (1:2,000; Santa Cruz USA), rabbit anti‐peroxisome proliferator‐activated receptor‐gamma coactivator‐1alpha (PGC1α) (1:3,000; Santa Cruz), rabbit anti‐nuclear factor erythroid 2‐related factor 2 (Nrf2) (1:3,000; Santa Cruz), rabbit anticatalase (1:5,000; Abcam UK), mouse anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (1:5,000; Abcam), and antiactin (1:5,000; Abcam).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Mutant SOD1 Increases APP Expression and Phosphorylation in Cellular and Animal Models of ALS

doi: 10.1371/journal.pone.0143420

Figure Lengend Snippet: Spinal cords from 30 -and 80-day-old and end stage SOD1 G93A mice were homogenized and their membrane fractions were subjected to immunoblot analysis. Age matched NT littermates served as control. For APP detection 22C11 antibody was used. For detection of phosphorylated APP (pAPP) a specific polyclonal anti pAPP T688 was used. β-Actin was used as an internal loading control. Immunoblot blot and densitometric analysis of the relative expression and phosphorylation levels of APP in the spinal cord of (A) 30-day-old mice, (B) 80-day-old and end stage SOD1G93A mice. All values are expressed as the mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey–Kramer post hoc test. N(NT 30D) = 3, N(G93A 30D) = 3, N(NT = 4), N(G93A 80D) = 4, N(G93A end stage = 3).*p < 0.05; **p < 0.01 (v.s. NT littermates).

Article Snippet: Protein concentration was determined using BCA protein assay kit (Thermo, USA), and samples were analyzed by immunoblotting with the following primary antibodies: mouse anti-APP 22C11 (1:2000 Millipore), rabbit anti-Phospho-APP T668 (1:1000 Cell Signaling Technology), rabbit anti-sAPPβ (1:500 Covance), anti-BACE C-terminus clone 61-3E7 (Millipore), PAb-240 mouse anti p53 (1:1000 Abcam), and SD-G6 mouse anti-human SOD1 (1:1000 Sigma), mouse anti βActin (1:10000 sigma), mouse anti Tubulin (1:10000 sigma) and Mouse anti-HA antibody (1:1000 Santa Cruz).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Mutant SOD1 Increases APP Expression and Phosphorylation in Cellular and Animal Models of ALS

doi: 10.1371/journal.pone.0143420

Figure Lengend Snippet: Soluble and membrane fractions from 80-day-old and end stage SOD1 G93A mice were subjected to immunoblot analysis. Age matched NT littermates served as control. A. sAPPβ was detected using polyclonal antibody specific for the neoepitope generated after BACE cleavage in the spinal cords soluble fractions. The levels of sAPPβ were normalized to β-Actin levels. B. Detection of BACE1 in the spinal cord homogenates membrane fraction was performed using a specific anti C terminal MAb. Spinal cord BACE1 levels were normalized to β-Actin levels. All values are expressed as the mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey–Kramer post hoc test. N(NT = 4), N(G93A 80D) = 4, N(G93A Final stage = 3).*p < 0.05; **p < 0.01 (v.s. NT littermates). CHO cells overexpressing APP 751 were transiently transfected with WT SOD1 or with mutant SOD1 G93A . C. Representative Western blot of mutant and WT SOD1 expression in CHO cells as a result of transient transfection. Human HA -tagged SOD1 was detected using anti HA antibody. Non-tranfected cells served as negative control D. Aβ42 peptides levels were measured in the medium using sandwich ELISA and the ratio between secreted Aβ42 in each treatment group and the untreated group was calculated and presented in percentage. The experiment was repeated three times. Statistical comparisons were performed using Student's t-test. *p value <0.05.

Article Snippet: Protein concentration was determined using BCA protein assay kit (Thermo, USA), and samples were analyzed by immunoblotting with the following primary antibodies: mouse anti-APP 22C11 (1:2000 Millipore), rabbit anti-Phospho-APP T668 (1:1000 Cell Signaling Technology), rabbit anti-sAPPβ (1:500 Covance), anti-BACE C-terminus clone 61-3E7 (Millipore), PAb-240 mouse anti p53 (1:1000 Abcam), and SD-G6 mouse anti-human SOD1 (1:1000 Sigma), mouse anti βActin (1:10000 sigma), mouse anti Tubulin (1:10000 sigma) and Mouse anti-HA antibody (1:1000 Santa Cruz).

Techniques: Western Blot, Generated, Transfection, Mutagenesis, Expressing, Negative Control, Sandwich ELISA

Journal: PLoS ONE

Article Title: Mutant SOD1 Increases APP Expression and Phosphorylation in Cellular and Animal Models of ALS

doi: 10.1371/journal.pone.0143420

Figure Lengend Snippet: Primary astroglial cells were isolated from brains of mutant SOD1 G93A pups. After 7 days in culture cells were treated with 13nM BBS for 24h. Induction of mutant SOD1 G93A fused to GFP expression in NSC34 stable line was performed by incubating the cells with Doxycycline for 48h. 24h after the induction the cells were treated with 26nM BBS for 24h. The levels of mutant SOD1, APP and actin were measured using Western blot analysis. SOD1 was detected by polyclonal anti human SOD1 antibody and APP was detected using 22C11 antibody. SOD1 levels were normalized to β-Actin levels. A. Levels of APP and SOD1 in primary astroglial cells. B. Levels of APP and SOD1 fused to GFP in NSC34 stable line. All values are expressed as the mean ± SEM. Statistical comparisons were performed using Student's t-test. *p < 0.05. For in vivo analysis of the effect of BBS, spinal cords of i.c.v. and i.p. treated SOD G93A mice were homogenized and subjected to immunoblot analysis. Analysis of the effect of BBS, in spinal cords of i.c.v. treated SOD G93A mice C. sAPPβ was detected in spinal cord soluble fractions and normalized to β-Actin levels. D. APP and SOD1 in spinal cords of i.c.v. treated mice were detected in membrane fractions and normalized to β-Actin. All values are expressed as the mean ± SEM. Statistical comparisons were performed using Student's t-test. N(BBS) = 4, N(Non relevant) = 5. *p < 0.05; **p < 0.01 (v.s. Non relevant). Analysis of the effect of BBS, in spinal cords of i.p. treated SODG93A mice. E. The levels of mutant SOD1, APP and tubulin in the membrane fraction were measured using Western blot analysis. Statistical comparisons were performed using Student's t-test. N(BBS) = 12, N(Control) = 11.*p < 0.05.

Article Snippet: Protein concentration was determined using BCA protein assay kit (Thermo, USA), and samples were analyzed by immunoblotting with the following primary antibodies: mouse anti-APP 22C11 (1:2000 Millipore), rabbit anti-Phospho-APP T668 (1:1000 Cell Signaling Technology), rabbit anti-sAPPβ (1:500 Covance), anti-BACE C-terminus clone 61-3E7 (Millipore), PAb-240 mouse anti p53 (1:1000 Abcam), and SD-G6 mouse anti-human SOD1 (1:1000 Sigma), mouse anti βActin (1:10000 sigma), mouse anti Tubulin (1:10000 sigma) and Mouse anti-HA antibody (1:1000 Santa Cruz).

Techniques: Isolation, Mutagenesis, Expressing, Western Blot, In Vivo

Journal: PLoS ONE

Article Title: Mutant SOD1 Increases APP Expression and Phosphorylation in Cellular and Animal Models of ALS

doi: 10.1371/journal.pone.0143420

Figure Lengend Snippet: A. Representative confocal images of boutons of a hippocampal neuron that was co-transfected with APP YFP and SOD1 CFP . Scale bar: 2 μm. B. Pseudocolor-coded fluorescence images of a bouton expressing APPYFP and SOD1CFP before and after YFP photobleaching, Scale bar: 2 μm. C. Summary of Em data for APPYFP / GB1aCFP (n = 26), APPYFP / SOD1CFP (n = 39), APPYFP / SOD1CFP (n = 42). Error bars represent SEM.

Article Snippet: Protein concentration was determined using BCA protein assay kit (Thermo, USA), and samples were analyzed by immunoblotting with the following primary antibodies: mouse anti-APP 22C11 (1:2000 Millipore), rabbit anti-Phospho-APP T668 (1:1000 Cell Signaling Technology), rabbit anti-sAPPβ (1:500 Covance), anti-BACE C-terminus clone 61-3E7 (Millipore), PAb-240 mouse anti p53 (1:1000 Abcam), and SD-G6 mouse anti-human SOD1 (1:1000 Sigma), mouse anti βActin (1:10000 sigma), mouse anti Tubulin (1:10000 sigma) and Mouse anti-HA antibody (1:1000 Santa Cruz).

Techniques: Transfection, Fluorescence, Expressing