Western blotting was performed with primary (rabbit) antibodies against LC3 (1:1,000; Abcam, Cambridge, MA, USA), Beclin-1 (1:1,000; CST, Danvers, MA, USA), p62 (1:1,000; CST), α-synuclein (1:1,000; Abcam) or β-actin (1:5,000; Applygen, Beijing, China). A fluorescently labeled secondary antibody (1:10,000; LI-COR Biosciences, Lincoln, NE, USA) was used.
α synuclein
Manufactured by Abcam
Sourced in United States, United Kingdom
α-synuclein is a protein that is primarily found in the presynaptic terminals of neurons. It is involved in the regulation of neurotransmitter release and the maintenance of synaptic function.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (3 mentions)
Phosphorylated α synuclein ser129, by Fujifilm (2 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Fluorescently labeled secondary antibody, by LI COR (1 mentions)
β actin, by Applygen (1 mentions)
Anti ikk, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Vector Laboratories (1 mentions)
Brain derived neurotrophic factor (bdnf), by Abcam (1 mentions)
β amyloid, by Abcam (1 mentions)
Bond max autostainer, by Leica (1 mentions)
Cd68 pgm 1, by Agilent Technologies (1 mentions)
Ventana discovery, by Roche (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Sartorius (1 mentions)
Psd 95, by Merck Group (1 mentions)
Tyrosine hydroxylase th, by Merck Group (1 mentions)
Horseradish peroxidase linked anti rabbit igg, by Cell Signaling Technology (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
15 protocols using α synuclein
1
Immunoblotting for Autophagy Markers
2
Hippocampal and Cortical Protein Studies
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Protein studies on the hippocampus and cortex were performed and quantified as described previously (Dubal et al., 2015 (link)). Additionally, α-synuclein (1:1,000, Abcam) and phosphorylated α-synuclein-Ser129 (1:1,000, Wako Pure Chemicals Industries) were measured. SDS (1%) was included in homogenates for optimal detection of the LMW GluN2B protein.
3
Subcellular Fractionation and Protein Profiling of Primary Microglia
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For the subcellular fractions, primary microglia-enriched cultures were lysed in hypotonic lysis buffer and incubated on ice for 30 min, and then subjected to homogenization. The lysates were loaded onto a sucrose gradient in lysis buffer, and the supernatant above the sucrose gradient was performed as the cytosolic fraction. The pellet was solubilized in hypotonic lysis buffer and was applied as the membranous fraction. For the whole cell lysate extraction, cultures were washed with cold phosphate-buffered saline (PBS) and lysed with cell lysis buffer. The lysates were incubated on ice for 30 min and then centrifuged at 12,000×g for 25 min. Protein levels were quantified via bicinchoninic acid (BCA) assay. Membranes were blocked with 5% non-fat milk and then incubated with the following primary antibodies: β-actin, TH, ionized calcium-binding adapter molecule-1 (Iba-1), α-synuclein, p47, p67, gp91, BDNF, GDNF (Abcam, Cambridge, UK), phospho-p65 (p-p65), p65, phospho-IKK (p-IKK), anti-IKK, phospho-p38 (p-p38), p38, phospho-JNK (p-JNK), JNK (Cell Signaling Technology, MA, USA), and horseradish peroxidase-conjugated secondary antibodies (Vector Laboratories, CA, USA). All the antibodies were diluted between 1:200 and 1:1000. The blots were developed with the enhanced ECL reagent.
4
Neuropathological Evaluation of Brain Tissue
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Formalin-fixed, paraffin-embedded brain and spinal cord tissue was available from selected regions (table 1 ). The paraffin sections were cut at 5 μm, mounted on glass slides, and stained with routine hematoxylin and eosin. Representative 5-μm sections were immunostained for TDP43, β-amyloid, α-synuclein, hyperphosphorylated tau, p62, and CD68 with the following antibodies: TDP43 (2E2-D3, 1:3,000, Abcam, Cambridge, UK), β-amyloid (6F3D, 1:50, DAKO, Glostrup, Denmark), α-synuclein (KM51, 1:50, Leica/Novocastra, Buffalo Grove, IL), AT8, (MN1020, 1:100, Invitrogen, Carlsbad, CA), p62 (3/P62LCK Ligand, 1:100, BD Transduction, East Rutherford, NJ), and CD68 (PG-M1, 1:100, DAKO), respectively. Immunostaining was performed on either a BondMax autostainer (Leica Microsystems, Wetzlar, Germany) or a Roche (Basel, Switzerland) Ventana Discovery automated staining platform following the manufacturer's guidelines, using biotinylated secondary antibodies and a horseradish peroxidase–conjugated streptavidin complex and diaminobenzidine as a chromogen. All immunostainings were carried with appropriate controls. Gliosis, microglial activity, and the density of TDP43 pathologic inclusions were scored semiquantitatively.
5
Neuronal Protein Expression Analysis
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Equal amounts of protein were separated by 4–12% Bis-Tris-polyacrylamide electrophoresis gel and transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary antibody Neu-N, PSD-95, tyrosine hydroxylase (TH, EMD Millipore, Temecula, CA, USA), α-synuclein, signal transducers (Abcam, Cambridge, MA,USA) and activators of transcription 1 (STAT1), p-p65, p65, p-IκBα and IκBα (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C and followed by horseradish peroxidase-linked anti-rabbit IgG (1:3000) for 2 h at 25 °C. ECL reagents (Biological Industries, Cromwell, CT, USA) were used as a detection system.
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Paraquat-Induced Neuroinflammation Model
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Paraquat, maneb and taurine were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). RNAiso Plus and SYBR Premix Ex TaqTM II were obtained from Takara Bio Inc. (Takara, Tokyo, Japan). The membrane protein extraction kit was obtained from Beyotime (Jiangsu, China). The following primary antibodies were used: TH (EMD Millipore Corporation, Billerica, MA, USA), α-synuclein (Abcam, Cambridge, MA, USA), Iba-1 (Wako Chemicals, Richmond, VA, USA), CD11b (AbD Serotec, Raleigh, NC, USA), phosphorylated p65, p65, phosphorylated IκBα, IκBα, phosphorylated IKKα (Cell Signaling Technology, Danvers, MA, USA), 4-HNE (Abcam, Cambridge, MA, USA), p47phox (EMD Millipore, Temecula, CA, USA), gp91phox (BD Transduction Laboratories, San Jose, CA, USA) and GAPDH (Abcam, Cambridge, MA, USA). The BCA Protein Assay Kit was purchased from Life Technologies (Waltham, MA USA). All other chemicals were of the highest grade commercially available.
7
Protein Expression Analysis in Parkinson's Disease
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We extracted the proteins in the fourth week after the success of the PD model. Proteins were extracted from tissues and cells with radioimmunoprecipitation assay lysis buffer (Beyotime, China), and the concentration of proteins was determined with bicinchoninic acid protein assay kit (Beyotime, China) according to the manufacturer’s instructions. Approximately 50 μg of protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Bedford, MA, USA). The membranes were blocked in quick block solution (Beyotime, China) for 15 min at room temperature and subsequently incubated with primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:3,000; Proteintech, China), α-synuclein (1:1,000; Abcam, UK), tyrosine hydroxylase (TH), α-synuclein, Iba-1, IL-1β, IL-6, Nrf2 and NQO-1 (1:1,000; Proteintech), and TNF-α (Affinity, China) overnight at 4°C. The membranes were washed with tris-buffered saline with 0.1% Tween 20 and incubated with HRP-conjugated secondary antibody (Proteintech) for 1 h at room temperature. The membranes were washed again and detected using a chemiluminescence western detection system (Bio-Rad, Hercules, CA, USA). Each experiment was conducted three times, and then calculated the mean values.
8
Protein Expression Analysis in Neuronal Tissues
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The protein expressions in SN tissues or PC12 cells were analyzed by Western blot analysis. The brain was removed under anesthesia. Each brain was dissected on a cold glass plate to separate the SN tissues. The SN tissues or PC12 cells pellets were homogenized in RIPA lysis buffer. The lysates were incubated on ice for 30 min, then centrifuged at 12,000 g for 15 min. The supernatant was then collected for the analysis of protein expressions. Determination of protein concentrations was carried out by BCA kit (Beyotime, Beijing, China). The protein was separated on 10% Bis-Tris NuPAGE gel and transferred to PVDF membrane. The PVDF membrane was blocked with 4% BSA (Sigma) for 2 h, and then reacted with primary antibodies at 4℃ for overnight. The primary antibodies included those for α-synuclein (1:1000, Abcam), LC3-I/II (1:1000, Abcam), Beclin-1 (1:1000, Cell signaling), SQSTTM1/P6
2 (1:1000, Cell signaling), mTOR (1:1000, Cell signaling), Phospho-mTOR (1:1000, Cell signaling), and β-actin (1:2000, Beyotime). After washing, the membranes were then incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) for 2 h. The membrane-bound secondary antibody was detected with ECL Western blot detection kit. The band intensities were quantified using Quantity One 1-D analysis software v4.52 (BioRad).
2 (1:1000, Cell signaling), mTOR (1:1000, Cell signaling), Phospho-mTOR (1:1000, Cell signaling), and β-actin (1:2000, Beyotime). After washing, the membranes were then incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) for 2 h. The membrane-bound secondary antibody was detected with ECL Western blot detection kit. The band intensities were quantified using Quantity One 1-D analysis software v4.52 (BioRad).
9
Acrolein-induced Neurodegeneration in Rats
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Forty-eight hours after intranigral infusion of acrolein, rats were deeply anesthetized and then perfused transcardially using 4% paraformaldehyde in 0.1 M PBS. Brains were removed and placed in 30% sucrose-PBS overnight and frozen-sectioned coronally at 30 μm thickness. Sections were washed with 0.1 M PBS, incubated with 0.3% Triton X-100 and 1% goat serum (GS; Sigma, St. Louis, MO), and blocked with 3% GS for 60 min. Sections were then incubated overnight at 4 °C with primary antibodies specific for TH (Cell Signaling Tech., Danvers, MA), α-synuclein (Abcam, Cambridge, UK) and FDP-lysine (Abcam, Cambridge, UK). Afterwards, sections were incubated for 1 h at room temperature with secondary antibodies conjugated with rhodamine and fluorescein isothiocyanate (Millipore Corporation, Billerica, MA,). Nuclei were labeled with 4′, 6-diamidino-2-phenylindole (1 mg/ml) for 10 min at room temperature. Sections were mounted in glycerol and visualized by a fluorescence confocal microscope (FluoView, Olympus, Tokyo, Japan). TH-positive cells in acrolein-infused SN were counted manually and expressed as % of that in the contralateral intact SN.
10
Hippocampal and Cortical Protein Studies
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