AHB0261

antibody from Invitrogen Antibodies

Targeting: SNCA NACP, PARK1, PARK4, PD1

Provider product page for AHB0261

Western blot

ELISA

Immunocytochemistry

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [22]
Comments [0]
Validations
Immunocytochemistry [1]
Flow cytometry [1]
Other assay [20]

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Product number: AHB0261 - Provider product page
Provider: Invitrogen Antibodies
Product name: alpha Synuclein Monoclonal Antibody (Syn 211)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: A suggested positive control for this product is SHSY-5Y cells. It will not cross-react with mouse or rat.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: Syn 211
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C

SNCA protein structure - AHB0261 shown in red.

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 p62- and Ubi-1 positive aggregates co-localize with IbA1-positive microglia. a - c Representative confocal microscopy images of p62 (green, second column ) and MAP2 ( a , red, first row ), Olig2 ( b , gray, second row ), or GFAP ( c , red, third row ) show no co-localization of p62 and the different cellular markers. In contrast, co-staining p62 (green) and IbA1 ( d , gray, fourth row ) or Ubi-1 (green) and IbA1 ( e , gray, fifth row ) revealed clear co-localization. IbA1 positive microglial cells ( f , gray, sixth row ) and GFAP-positive astroglia ( g , red, seventh row ) also co-stained for aSYN ( arrows in f , g ). Scale bar for a - g 25 mum

Supportive validation

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Experimental details: Figure 4 alpha-syn overexpression decreased striatal TH expression. A - B . Pseudocolored near infrared TH immunofluorescence in the alpha-syn overexpressing (lesion) and intact striatum of 1.0 x 10 13 (vg/ml) titer ( A ) and 1.0 x 10 14 (vg/ml) titer ( B ) injected rats. C . TH expression was significantly reduced in the alpha-syn overexpressing striatum (*p < 0.001).

Supportive validation

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Experimental details: Figure 1 DJ-1 inhibits alpha-synuclein accumulation in SH-SY5Y cells. (A-C) DJ-1 deficiency aggravated the accumulation of alpha-synuclein in SH-SY5Y cells. Wild-type (WT) alpha-synuclein plasmid and DJ-1 siRNA were co-transfected into SH-SY5Y cells for 48 h. Immunoblots for the indicated proteins are shown in (A) , and quantifications of changes in the levels of alpha-synuclein and DJ-1 are shown in (B,C) respectively (mean +- SEM, n = 3, *** p < 0.001 vs. alpha-synuclein vector control, ### p < 0.001 vs. scrambled control). (D-F) WT DJ-1 overexpression inhibited accumulation of alpha-synuclein in SH-SY5Y cells. WT alpha-synuclein plasmid and WT DJ-1 plasmid were co-transfected into SH-SY5Y cells for 48 h. Immunoblots for the indicated proteins are shown in (D) , and quantifications of changes in the levels of alpha-synuclein and DJ-1 are shown in (E,F) respectively (mean +- SEM, n = 6, *** p < 0.001 vs. alpha-synuclein vector control, ### p < 0.001 vs. DJ-1 vector control).

Supportive validation

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Experimental details: Figure 2 DJ-1 inhibits the accumulation and aggregation of alpha-synuclein in Parkinson's disease (PD) animal models. (A-H) DJ-1 deficiency accelerates the accumulation and aggregation of alpha-synuclein in DJ-1-/- mouse ventral midbrain samples after Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in the SDS-soluble and urea-soluble fraction compared with that of DJ-1+/+ mice. Immunoblots for alpha-synuclein are shown in (A) for TBS-soluble, (C) for TX-100-soluble, (E) for SDS-soluble and (G) for urea-soluble fractions. In addition, quantifications of changes in alpha-synuclein levels are shown in (B,D , F,H) (mean +- SEM, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. saline control; # p < 0.05 vs. DJ-1+/+).

Supportive validation

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Experimental details: Figure 3 Endogenous alpha-synuclein is degraded in lysosomes by chaperone-mediated autophagy (CMA) in SH-SY5Y cells. (A,B) Levels of endogenous alpha-synuclein were determined in SH-SY5Y cells treated with NH 4 Cl for 9.5 h. Immunoblots for the indicated proteins are shown in (A) and quantification of alpha-synuclein levels is shown in ( B ; mean +- SEM, n = 3, ** p < 0.01 vs. control). (C-H) Levels of endogenous alpha-synuclein were determined in SH-SY5Y cells treated with Chloroquine diphosphate (CQ), 3-methyladenine (3-MA) and lactacystin respectively for 24 h. Immunoblots for the indicated proteins are shown in (C , E,G) , and quantifications of alpha-synuclein levels are shown in (D , F , H) respectively (mean +- SEM, n = 3, ** p < 0.01 vs. control). (I-K) Levels of endogenous alpha-synuclein were determined in lysosome-associated membrane protein type-2 (LAMP2) knockdown SH-SY5Y cells. Immunoblots for the indicated proteins are shown in (I) , and the quantifications of alpha-synuclein and LAMP2A levels are shown in (J,K) respectively (mean +- SEM, n = 3, *** p < 0.001 vs. control, ### p < 0.001 vs. scramble). (L-N) Levels of endogenous alpha-synuclein were determined in LAMP1 knockdown SH-SY5Y cells. Immunoblots for the indicated proteins are shown in (L) , and the quantifications of alpha-synuclein and LAMP1 levels are shown in (M,N) respectively (mean +- SEM, n = 3, *** p < 0.001 vs. control, ### p < 0.001 vs. scramble).

Supportive validation

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Experimental details: Figure 4 Effects of DJ-1 deficiency on CMA and alpha-synuclein levels in SH-SY5Y cells. (A-D) Levels of LAMP2A, HSC70, LAMP1, alpha-synuclein and DJ-1 were determined in homogenates of SH-SY5Y cells co-transfected with WT alpha-synuclein plasmid and DJ-1 siRNA. Immunoblots for the indicated proteins are shown in (A) , and quantification of the levels of LAMP2A, HSC70 and LAMP-1 are shown in (B-D) respectively (mean +- SEM, n = 3, 3, 4, ** p < 0.01 vs. vector control, ## p < 0.01 vs. scramble). (E-H) Levels of LAMP2A, HSC70 and LAMP-1 in lysosome-enriched fractions isolated from SH-SY5Y cells co-transfected with WT alpha-synuclein plasmid and DJ-1 siRNA were determined. Immunoblots for the indicated proteins are shown in (E,G) , and quantifications of the levels of LAMP2A and HSC70 are shown in (F,H) respectively (mean +- SEM, n = 4, * p < 0.001 vs. vector control, ## p < 0.01 vs. scramble).

Supportive validation

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Experimental details: Fig. 1 AAV8-mediated human WT alphaSyn expression in the SN and alphaSyn oligomerization detected by BiFC in mice at 4 weeks post-AAV injection. (a) Schematic structures of venus and alphaSyn-BiFCs (V1S and SV2). Upper right illustrates reconstitution of venusYFP fluorescence by alphasyn-alphasyn interactions. (b) TH immunofluorescence and venusYFP fluorescence in the SN. Tissue sections were counterstained with DAPI (blue). Scale bar: 30 mum. (c) Immunohistochemistry of human alphaSyn in the SN after V1SSV2 injection. Tissue sections were counterstained with hematoxylin (blue). Scale bars: 100 mum (left), 10 mum (right). (d) immunofluorescent double staining for TH and human alphaSyn and their colocalization with venusYFP in the SN after V1SSV2 injection. (e) VenusYFP before and after proteinase K treatment (PK- and PK+) in the SN following V1SSV2 injection. Scale bar: 30 mum. Fig. 1

Supportive validation

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Experimental details: Fig. 2 Human alphaSyn transduction induces synucleinopathies. (a) VenusYFP fluorescence in the striatum, venusYFP fluorescence and its colacalization with TH in the SN 8 weeks following venus or V1SSV2 injection. Arrows indicate beaded, dystrophic neurites. Scale bars: 100 mum (left), 10 mum (right). (b) VenusYFP before and after proteinase K treatment in the SN 12 weeks following V1SSV2 injection. Scale bars: 20 mum. (c) Immunohistochemistry for pSer129 alphaSyn in the SN 12 weeks following V1SSV2 injection. Scale bars: 50 mum (left), 10 mum (right). (d) Accumulation of high-molecular-weight-alphaSyn species in the ventral midbrain and the striatum 8 weeks following V1SSV2 injection by Western blot analysis using anti-human alphaSyn antibody. Tissue sequential extractions were prepared using Triton X-100 and SDS. Fig. 2

Supportive validation

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Experimental details: Fig. 5 alpha-Synuclein inclusions in MSA-infected astrocytes are ubiquitinated and co-localized with p62 or contain alpha-synuclein phosphorylated at Y125. Representative immunographs of TgM83 +/+ primary astrocytes exposed to 0.5% secondary passaged MSA 2 brain homogenate for 48 h and immunostained at 0, 7, 14, and 21 days post-exposure (dpe) for a ubiquitin (green) and pSyn (S129) (red), b p62 (green) and pSyn (S129) (red), and c alphaSyn (green) and pSyn Y125 (magenta). Merge channels are shown, and c insets of dashed areas are shown in the bottom row. Nuclei were stained with DAPI (blue). Scale bars, 20 mum

Supportive validation

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Experimental details: Fig. 2. Neutral lipids accumulate in PD patient brains relative to age-matched healthy subjects and localize to aSYN-rich inclusion bodies within dopaminergic neurons of the SN. ( A ) Fluorescent micrographs labeled for neutral lipids (BODIPY) (gray-scale) in neuromelanized dopaminergic neurons of the HS SN and PD SN. Neuropigmented cell profiles were manually isolated from the surroundings for clarity. ( B ) Representative micrographs of neutral lipids (BODIPY; green) and TH (magenta) colabeled neurons in HS SN and PD SN. Nuclei (TO-PRO; blue) are shown merged. The dotted line represents TH + cell outlines. Boxed insets are shown in higher magnification with orthogonal views on the right (scale bar: 25 um). ( C ) Automated overlap coefficients of images colabeled as in B of the BODIPY + /TH + area over the total BODIPY + area, expressed as lipid (fraction overlap). * P < 0.05, n = 5 to 6 subject averages per group, n = 3 to 5 fields of view containing one or two cells per field (Z-stacks) per subject. The line indicates the mean, with individual values shown. ( D ) Representative micrograph of neutral lipid (green) and aSYN (magenta) in HS SN and PD SN. The dotted outline represents neuromelanin + cell profiles as identified by bright-field microscopy before confocal imaging. Boxed insets are shown in higher magnification with orthogonal views on the right. Filled arrowheads indicate intracellular Lewy bodies; empty arrowheads indicate Lewy body-like inclusions not associate

Supportive validation

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Experimental details: FIGURE 5 miR-23b-3p, miR-30b-5p, and miR-195-3p coordinately regulate downstream PD-related target genes in NSCs. (A) Diagram showing the direct targeting of miR-23b-3p on the UTR of SNCA. (B) The mRNA levels of SNCA were examined in NSCs transfected with miR-23b-3p mimic (mimic-23b-3p) or inhibitor (inhibitor-23b-3p) by quantitative PCR. (C) The protein level of SNCA was examined in NSCs transfected with miR-23b-3p mimic (mimic-23b-3p) or miR-23b-3p inhibitor (inhibitor-23b-3p) by Western blotting using antibody against SNCA. (D) The luciferase activity of HEK293 cells transfected with pMiRluc-SNCA or pMiRluc-SNCAM (mutation in the miRNA targeting site) together with mimic-control (pMiRluc-SNCA) or mimic-23b-3p (pMiRluc-SNCA+mimic). After incubation overnight, the relative luciferase activity was measured and normalized to the Renilla activity. ***means statistically P

Supportive validation

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Experimental details: Figure 4 BK50118-C significantly reduced alpha-synuclein but not p-tau levels in TgA53T mice. Male and female TgA53T mice were treated with intraperitoneal injection of vehicle or BK50118-C at the daily dosage of 10 mg/Kg or 40 mg/Kg for 7 days. WB of midbrain lysates showing ( A ) the levels of alpha-synuclein relative to actin on 4-12% SDS-NuPAGE gel in the above mice. ( B ) The first blot is the WB densitometry. The 2nd and 3rd blots are IP alpha-synuclein (syn) or ubiquitin. ELISA showing ( C ) the levels of alpha-synuclein and ( D ) ptau396 following BK50118-C treatment. Asterisks indicate statistic significant difference * p < 0.05 or ** p < 0.01 vs. corresponding vehicle. Ordinary one-way ANOVA was used for the analysis. N = 3-4 mice per group. DAB +Nissl staining. C significantly reduced alpha-synuclein levels in cortex ( F / H ) and striatum ( K / M ) compared to the corresponding vehicles ( E / G ) or ( J / L ) , verified by the quantification of alpha-synuclein positive cells in cortex ( I ) and striatum ( N ). Two-tailed student t test was used for analysis, and * indicates p < 0.05 vs. vehicle. N = 3-4 per group. All values presented as Mean +- SD. Scale bars: 100 um ( E , F , J , K ) or 200 um ( G , H , L , M ).

Supportive validation

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Experimental details: Figure 5 BK50118-C significantly increased alpha-synuclein ubiquitination but had minimal effects on tyrosine hydroxylase (TH) levels in striatum of TgA53T mice. Male and female TgA53T mice were treated with intraperitoneal injection of vehicle or BK50118-C at the daily dosage of 40 mg/Kg for 7 days. Immunochemistry assay of 20 um thick brain sections showed alpha-synuclein (green), ubiquitination (red) and DAPI (blue) staining in the striatum of TgA53T mice treated with either DMSO ( A , C , E ) or BK50118-C ( B , D , F ). BK50118-C increased alpha-synuclein ubiquitination, verified by optic density of co-localization ( G ). Immunostaining showed TH (red) and DAPI (blue) staining in vehicle DMSO ( H , J , L ) and BK50118-C ( I , K , M ). BK50118-C had minimal effects on TH levels in striatum, verified by quantification of optic density ( N ). Asterisk indicates statistic significant difference * p < 0.05; two-tailed student's t test was used for analysis. N = 3-4 mice per group. All values presented as Mean +- SD. Scale bars: 200 um.

Supportive validation

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Experimental details: Figure 5 BK50118-C reduces alpha-synuclein and protects against dopamine loss in USP13 +/+ , but not USP +/- or USP13 -/- brain. WB of STEN (soluble) midbrain extracts of ( A ) USP13 +/+ , USP +/- and USP13 -/- mice, which were injected with lentiviral alpha-synuclein into SN and treated with BK50118-C or DMSO showing alpha-synuclein (17 kD) level relative to actin (42 kD) on 4-12% Criterion(tm) XT Bis-Tris Protein Gel, and ( B ) alpha-synuclein densitometry. ELISA measuring levels of ( C ) human alpha-synuclein ( D ) dopamine, and ( E ) HVA in same brain extracts. The asterisk indicates statistically significant difference. One-way ANOVA ** p < 0.01. Two tailed Student's t test, and non parametric tests # p < 0.05. n = 3-6 per group. Mean +- SD. The black dot * represents each individual data.