32-8100

antibody from Invitrogen Antibodies

Targeting: SNCA NACP, PARK1, PARK4, PD1

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Antibody Data
Antigen structure
References [32]
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Western blot [2]
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Product number: 32-8100 - Provider product page
Provider: Invitrogen Antibodies
Product name: alpha Synuclein Monoclonal Antibody (Syn 211)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: This antibody reacts with the alpha-synuclein protein. This antibody does not cross-react with beta- or gamma-synuclein or synoretin. During development reactivity was confirmed with human. Reactivity with other species has not been tested.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: Syn 211
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

SNCA protein structure - 32-8100 shown in red.

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Experimental details: NULL

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Experimental details: NULL

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Experimental details: NULL

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Experimental details: NULL

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Experimental details: Figure 1. alpha-Synuclein sumoylation in vitro in cells and in mouse brain. (A) alpha-Synuclein is SUMO2 modified in vitro. 500 ng alpha-synuclein, 500 ng SUMO2, 150 ng Aos1/Uba2, 200 ng Ubc9, and 5-10 ng E3 ligase fragments were incubated for 30 min at 30degC with and without ATP in a volume of 20 ul. Reactions were stopped with SDS sample buffer before analysis by SDS-PAGE and immunoblotting with mouse monoclonal anti-alpha-synuclein antibody (Syn211; Invitrogen). (B) alpha-Synuclein modification by SUMO2 in HEK293T cells. Plasmids encoding His 6 -SUMO2 (His 6 -S2) or/and alpha-synuclein were transfected in HEK293T cells. SUMO substrates were purified by Ni 2+ affinity chromatography (Ni 2+ -NTA) under denaturing conditions, and a sumoylated alpha-synuclein band at ~35 kD (stars in A and B) was detected with an alpha-synuclein antibody (Syn211). 1% of total input (TP) and 25% of elution fractions were loaded. (C, top) A schematic representation of the Thy1.2/His 6 -SUMO transgene. (bottom) alpha-Synuclein sumoylation in mouse brain tissue. Total His 6 -SUMO2-conjugated proteins were isolated using Ni 2+ -NTA affinity chromatography. TBLs and eluates were probed with anti-alpha-synuclein antibody recognizing mouse alpha-synuclein (clone 42). alpha-Synuclein modified by a single SUMO2 molecule, indicated with a star, could be detected in the eluate obtained from His 6 -SUMO2 transgenics.

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Experimental details: Figure 4. Sumoylation-impaired alpha-synuclein mutants (2KR and 2A) induce increased toxicity and inclusion formation compared with WT alpha-synuclein in HEK cells. (A-D) HEK293T cells were transfected with bicistronic plasmids expressing alpha-synuclein variants (syn-WT; 2KR: K96 and 102R; and 2A: D98A and E104A) with a PDZ binding tag (HSTTRV) and PDZ domain-EGFP fusion protein (GFP). (A-C) Cells were classified in three groups based on GFP fluorescence distribution: homogenous (A), containing intracellular GFP-positive inclusions (B, arrowheads), and preapoptotic or having fragmented nuclei by DAPI staining (C). Images display exemplary cells to illustrate the different categories into which cells were grouped. Because individual cells displayed variations in expression levels, exposure times were adjusted differently to avoid overexposure. At the level of total cell lysates, expression levels of the different constructs were the same between the different experimental conditions. Bar, 20 um. (D) Bar graphs represent percentages of all GFP-positive cells in the three categories summarized as mean +- SEM. Comparisons were made with KyPlot 5.0 using Student's t test (*, P < 0.05; **, P < 0.01). n = 6. Note that cell counts were performed by direct observation at the microscope using identical optical parameters for all experimental conditions. ns, not significant.

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Experimental details: Figure 1 Overexpression of alpha Syn variants in PMN. ( a ) Vector maps of the plasmids used to overexpress EGFP, alpha Syn-WT, -A30P and -A53T. The respective transcripts are expressed under the control of a human synapsin-1 promoter. ITR: AAV-2 inverted terminal repeat. Int: intron. SV40-pA: SV40 polyadenylation site. WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. bGH-pA: bovine growth hormone polyadenylation site. ( b and c ) Immunoblots of whole cell protein lysates from PMN transfected with the given plasmids (cells were lysed on DIV 5). In b , an antibody specific for human alpha Syn (LB509, Invitrogen) was used to detect only the alpha Syn expressed by the plasmids. In c , total cellular alpha Syn levels were assessed using an antibody recognizing both human and rat alpha Syn (BD). At the bottom, quantifications of the band intensities normalized to beta -tubulin are shown ( n =3; error bars represent means+-S.E.M.; ** P

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Experimental details: Figure 5 Colocalization of LC3 and alpha Syn in PMN transfected with different alpha Syn variants. ( a ) Representative confocal micrographs of PMN transfected with the respective alpha Syn variant given on the left side and immunostained against TH, LC3 and alpha Syn as specified on top. ( b ) Quantification of colocalization between alpha Syn and LC3 in the soma of TH-positive neurons after transfection with different alpha Syn variants represented by means of Li's intensity correlation coefficient. Cells transfected with alpha Syn-A30P show significantly less colocalization than those transfected with alpha Syn-WT or -A53T. Statistics: one-way ANOVA followed by Tukey-Kramer post hoc test, *** P

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Experimental details: Figure 6 Axonal transport of synaptophysin-positive vesicles in PMN transfected with different alpha Syn variants. ( a ) Representative micrograph showing PMN transduced with AAV.synaptophysin-EGFP (expressing EGFP-tagged synaptophysin) and co-transfected with p. alpha Syn-WT and p.dsRed (to identify transfected neurons) (DIV 5). In the higher magnification pictures, the EGFP-positive vesicles (arrows) can be seen along the transfected axon. ( b ) Representative kymographs of the movements of EGFP-tagged synaptophysin along neurites transduced with the plasmids given on the left side within 10 s ( y axis). Arrows point at representative moving vesicles, asterisks mark stationary vesicles. ( c-f ) Quantifications of synaptophysin-EGFP transport in neurites transfected with the given plasmids. Statistics: one-way ANOVA followed by Dunnett's post hoc test, * P

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Experimental details: Fig. 5 Stereological assessments of brain pathology and integrity. Representative images of microglial (CD68; a + b ), phosphorylated-tau ( c + d ), TDP-43 ( e + f ), ubiquitin ( g + h ) and alpha-synuclein ( i + j ) staining in the hippocampus (CA1) from LBC1936 ( a , c , e , g , i ) and AD ( b , d , f , h , j ) brains. Large scale bar = 50 mum, insert scale bars = 10 mum. k Histogram showing neuronal densities in eight cortical regions from the LBC1936 (black bars) and AD (grey bars) brains. l Histogram showing microglial densities (CD68-positive cells) in eight cortical regions from the LBC1936 and AD brains. m Histogram of cortical thickness measurements from eight cortical regions in the LBC1936 and AD brains

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Experimental details: Fig. 7 Using synaptoneurosomes to enrich and analyze synaptic proteins by western blotting. a Representative enrichment blot showing the exclusion of nuclear histone (17 kDa) from the synaptoneurosome preparation and retention of synapsin (40-80 kDa). b GluN2B western blot from the LBC1936 (black bars) and AD (grey bars) preparations. To assess protein integrity and control for post-mortem degradation [ 52 ], band2 (black arrow; 150 kDa) was divided by band1 (grey arrow; 170 kDa) to generate a ratio, and a value >=1 (red dashed line) is achieved by all samples except the LBC1936 EC (asterix). c Representative synaptophysin (40 kDa) blot of LBC1936 synaptoneurosomes and AD samples. d Representative total tau (Tau13; 45-60 kDa) blot of LBC1936 samples and AD samples. e Phosphorylated-tau (PHF1; 50-65 kDa) blot shows almost exclusive expression in the AD samples compared to the LBC1936 synapses. f Representative VDAC (29-32 kDa) blot of LBC1936 and AD synaptoneurosomes. GAPDH (36 kDa) or beta-actin (42 kDa) was run as a loading control. Histogram represents the mean of three experimental repeats and the bars represent the quantification of the bands directly above

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Experimental details: Fig. 1 Lentivirus vectors over-expressing mutant neurosin reduce the accumulation and propagation of alpha-synuclein. Wild-type neurosin (LV-wt-NR), neurosin-apoB (LV-wt-NR-apoB), the point mutant neurosin (LV-NR-R80Q) and R80Q-apoB (LV-NR-R80Q-apoB) were cloned into the 3 rd generation lentivirus vector. a B103 neuronal cells were transduced with the vectors and examined by immunoblot for neurosin and the epitope tag V5. b B103 transduced cells were also immunostained for neurosin expression. c Compared to LV-Control, neuronal cells transduced with the neurosin constructs expressed higher levels of neurosin, levels were comparable between the wt and mutant neurosin constructs. d An in vitro neuronal co-culture system was devised to mimic the propagation of alpha-syn. B103 neuronal ""Donor cells"" infected with LV-alpha-syn or LV-Control (red) were plated in cell culture inserts containing a 0.4 mum membrane. B103 neuronal ""Acceptor cells"" infected with LV-NR-R80Q, LV-NR-R80Q-apoB or LV-Control were plated on coverslips. e Confocal microscopy and immunocytochemical analysis showing alpha-syn (red) from the Donor Cells was secreted and taken up by Acceptor cells that were double labeled with an antibody against neurosin (green). f Image analysis of double labeled neuronal cells, results are expressed as percent of acceptor cells containing alpha-syn immunoreactivity. * - indicates one way ANOVA with post hoc Dunnett's, p < 0.05 compared to LV-Control. # - indicates one

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Experimental details: Fig. 4 Delivery of LV-NR-R80Q-apoB reduces alpha-syn accumulation in MBP-alpha-syn tg mice. a Bright field microscopy analysis of serial longitudinal vibratome sections from the non-tg and MBP-alpha-syn tg mice immunostained with an antibody against total alpha-syn (syn-1) and analyzed for alpha-syn positive cells following treatment with LV-Control, or LV-NR-R80Q-apoB vector. b Computer aided image analysis for numbers of alpha-syn positive cells in the striatum and the corpus. c Samples that included the cortex, corpus callosum and striatum were fractioned by ultracentrifugation and analyzed by immunoblot analysis with an antibody against total alpha-syn (syn-1). d Densitometry analysis of the levels of alpha-syn immunoreactivity plotted against actin. e Laser scanning confocal microscopy of vibratome sections from the non-tg and MBP-alpha-syn tg immunostained with an antibody against human alpha-syn (syn211) and analyzed for alpha-syn positive cells following treatment with LV-Control, or LV-NR-R80Q-apoB vector. f Computer aided image analysis of the number of alpha-syn positive cells in the striatum and the corpus callosum. g Bright field microscopy analysis of serial longitudinal vibratome sections form the non-tg and MBP-alpha-syn tg immunostained with an antibody against phosphorylated alpha-syn (Ser129) and analyzed for alpha-syn positive cells following treatment with LV-Control, or LV-NR-R80Q-apoB vector. h Computer aided image analysis for numbers of phosphorylated

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Experimental details: Fig. 5 Delivery of LV-NR-R80Q-apoB reduces the propagation of alpha-syn to astrocytes and clearance via microglia in MBP-alpha-syn tg mice. Vibratome brain sections from the non-tg and MBP-alpha-syn tg that received i.p. injections of LV-Control or LV-NR-R80Q-apoB were double immunofluorescence labeled with antibodies against cellular markers and human alpha-syn and analyzed with the laser scanning confocal microscope with an optical image of 1 mum with fluorescent signals in co-registry. Dotted box to the left depicts the image field zoomed represented under detail. a Double immunolabeling for the astrocyte marker S100 (red) and human alpha-syn (green) with nuclei (DAPI, blue). Co-immunolabeling is represented by signal in yellow. b Computer aided image analysis of the % of S100 cells displaying alpha-syn immunofluorescence in the corpus callosum and striatum. c Double immunolabeling for the microglial marker Iba-1 (red) and human alpha-syn (green) with nuclei (DAPI, blue). Co-immunolabeling is represented by signal in yellow. d Computer aided image analysis of the % of Iba-1 cells displaying alpha-syn immunofluorescence in the corpus callosum and striatum. n = 10 mice per group 9-10 m/o at the end of the treatment. Scale bar = 10 mum, detail = 20 mum. # indicates statistical significance ( p < 0.05, one way ANOVA, post hoc Tukey-Kramer) compared to LV-Control treated MBP-alpha-syn tg mice

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Experimental details: Fig. 4 Hypoestoxide reduces deposition of alpha-synuclein in a mouse model of PD. a Representative immunohistochemical staining of alpha-synuclein in the frontal cortex, hippocampus, and striatum. b Optical density analysis for alpha-synuclein-positive neuropil in the frontal cortex ( n = 5 per each group; two-way ANOVA, Bonferroni's multiple comparison post-test; *** p < 0.001). Error bars represent +-SEM. c The numbers of alpha-synuclein-positive cells in the frontal cortex. d , e Optical density analysis of immunoreactivity of alpha-synuclein in the hippocampus ( d ) and in the striatum ( e ) ( n = 5 per each group; two-way ANOVA, Bonferroni's multiple comparison post-test; ** p < 0.01, *** p < 0.001). Error bars represent +-SEM. f , g Biochemical analysis of SDS-soluble ( f ) and SDS-insoluble ( g ) fractions from the frontal cortex for alpha-synuclein ( n = 5 per each group; two-way ANOVA, Bonferroni's multiple comparison post-test; ** p < 0.01, *** p < 0.001). Error bars represent +-SEM. Scale bars = 250 mum (low magnification) and 25 mum (high magnification)

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Experimental details: Fig. 5 Dot blot assay for aggregated human aSyn in A53T mice. The tissue samples were homogenized and incubated for 10 min with proteinase K to digest the soluble forms of aSyn. Samples were blotted and revealed through an antihuman aSyn antibody to visualize proteinase K-resistant fraction. Each row shows a scheme of the analyzed area, the quantification of n =5 blots and an exemplary blot for a . cortex; b . hippocampus; c . midbrain; and d . cerebellum

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Experimental details: Fig. 6 Histological evaluation of aSyn aggregation in A53T mice at disease onset using a p-S129-aSyn antibody for all the regions tested. This posttranslational modification was shown to be found in the aggregated form of aSyn only. Each row shows a scheme of the analyzed region, a quantification of p-S129-aSyn signal through fluorescence intensity ( n =5 animals for each group), and representative photomicrographs. a . CA3 region; b . cortex (CX); c . dentate gyrus (DG); d . deep mesencephalic reticular nucleus (DpMe); e . red nucleus (RN); f . substantia nigra (SN). Scale bar = 20 um

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Experimental details: Figure 5. Reduced sumoylation exacerbates alpha-synuclein neurotoxicity in vivo. (A) A schematic representation of vector genomes used in the in vivo rat model of PD. Recombinant adeno-associated viral vectors (AAV2) express either EGFP, WT alpha-synuclein (syn-WT), or alpha-synuclein K96R and K102R (syn-2KR) under the control of neuron-specific human synapsin 1 gene promoter (hSyn1). Vectors also encode inverted terminal repeats (TR) and small control elements woodchuck hepatitis virus posttranscriptional control element (WPRE) and bovine growth hormone (bGH)-derived polyadenylation site. (B) VMAT2-positive (VMAT2 + ) neurons (red) in the left SN. (C) rAAV2-mediated GFP transduction of SNpc. (D) Overlay of VMAT2 + and GFP-transduced neurons in the left SN. (E-G) Coimmunostaining of VMAT2 + (red) and human alpha-synuclein (green). Survival of VMAT2 + neurons in SNpc of animals injected with rAAV2 vectors expressing EGFP (E), human WT alpha-synuclein (F), or alpha-synuclein-2KR (G). Panels B-G are each a composite of two separate fluorescent images (Axioplan2 with a 5x objective and AxioVision 4.7). (H and I) Respective unbiased stereological quantification of surviving VMAT2 + (H) or NeuN + (I) cells in the transduced SNpc. The graphs display VMAT2 + or NeuN + cell numbers in animals injected with rAAV2 expressing EGFP, WT alpha-synuclein (syn-WT), and alpha-synuclein-2KR (syn-2KR). N.I., contralateral noninjected side. n = 6-8 per experimental group. Results are mean +- SEM.

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Experimental details: Figure 1 Representative example of alpha-synuclein and thioflavin S staining in cultured cells (A) Transfected SH-SY5Y cells expressing A53T alpha-synuclein (alpha-syn) were cultured as described (), fixed, and stained using alpha-syn syn211 (red) and Thio S (green). The microscopy setup included an Olympus IX-FLA using BP360-370 (DAPI), BP420-480 (Thio S), and BP510-550 (alpha-syn) exciter filters. (B) iPSC-derived midbrain neurons from controls or patients harboring the A53T alpha-synuclein mutation were cultured for 90 days, fixed, and stained using LB509 for alpha-syn (red) and Thio S (green), and nuclei were detected with DAPI (blue), using a similar setup as described in (A). (C) Representative images of Thio S stained alpha-synuclein positive neurites from A53T iPSC-derived neurons as described in (B). The scale bars are 10 mum in all images. Arrowheads indicate large juxtanuclear inclusions, while arrows indicate cells with smaller punctated aggregates and diffuse Thio S staining. Asterisks show examples of background/non-specific staining of dead cells.

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Experimental details: 10.1371/journal.pone.0262464.g001 Fig 1 Mercury in the locus ceruleus, substantia nigra, cerebral cortex and striatum in Parkinson's disease. ( A ) Black mercury grains (eg, open arrowheads) are present in the cytoplasm of most locus ceruleus neurons in PD1 (main image) and PD2 (inset). Lewy bodies with haloes (filled arrowheads) are seen in some mercury-containing neurons. Numerous oligodendrocytes have small mercury deposits (eg, arrows) adjacent to their nuclei. Autometallography/Luxol fast blue. ( B ) Magenta immunostaining of a-synuclein shows co-localisation of Lewy bodies (open arrowheads) with black-staining mercury in locus ceruleus neurons of PD1. An extra-neuronal a-synuclein aggregate (open arrow) has no associated mercury. No Lewy bodies are present in a locus ceruleus neuron not containing mercury (filled arrowhead). The right upper inset shows a nearby locus ceruleus neuron at higher magnification with Lewy body/mercury co-localisation. The left lower inset shows mercury within a Lewy body. Autometallography/a-synuclein Magenta/hematoxylin. ( C ) Brown DAB immunostaining of a-synuclein shows co-localisation of a Lewy body with mercury (filled arrow) in a remaining substantia nigra neuron of PD1. A nearby Lewy neurite (right upper inset) shows associated mercury grains (open arrowhead). Some intraneuronal (filled arrowhead) and extraneuronal (*) Lewy bodies do not appear to contain mercury, possibly because of masking by the dense brown DAB staining. Scattered o

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Experimental details: Fig. 2. Exogenous addition of alpha-syn oligomers induces the intracellular aggregation of alpha-syn in 2D SH-SY5Y cultures. (A) Double immunostaining of undifferentiated and differentiated SH-SY5Y cells reveals intracellular aggregation of alpha-syn following 24 h incubation with seeding oligomers. alpha-Syn inclusions in undifferentiated cells display several distinct, punctate accumulations dispersed throughout the cytoplasm, consistent with aggregates. In contrast, the RA+BDNF cell lines show a single prominent accumulation (red; arrows) and obscure the nucleus (asterisks). Scale bars: 50 um. (B) Quantification of inclusion area (reflected as um 2 ) shows inclusions that are present in differentiated cells to be much larger than those in undifferentiated cells (measurements obtained from three independent experiments; n= 50). All data points are present, with mean shown as a straight line. (C) The percentage of cells containing inclusions was determined by counting 100 cells per repeat. Cells were deemed to contain inclusions if aggregation matched that in reference images when compared to controls. Data points are presented from three independent biological repeats. (B,C) Differences between treatments were tested for significance using Kruskal-Wallis test with post-hoc Dwass-Steel-Chritchlow-Fligner for >6 data points and Conover-Inman for

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Experimental details: Fig. 5. alpha-Syn-seeded inclusions in 3D are indicative of in vivo Lewy body inclusions. alpha-Syn oligomer treatments to 3D-differentiated cultures develop alpha-syn-positive inclusions that demonstrate the same morphology as in vivo Lewy bodies (LBs; A) and are positive for the marker ubiquitin (B). Dotted line in purple highlights cell nuclei, whereas brown dotted line highlights stained area. (C) Immunofluorescence shows colocalisation of alpha-syn (red) and ubiquitin (green) staining. Nuclei were visualised using DAPI staining (blue). Scale bars: 50 um (left) and 10 um (right). In vivo pathology image displayed with permission from Springer Nature. This image is not published under the terms of the CC-BY license of this article. For permission to reuse, please see Chesselet et al. (2012) .