The samples of mitochondrial lysates (1 mg/mL) dissolved in Laemmli buffer (Bio-Rad, Hercules, CA, USA) were added to each line at 20 μg and divided by electrophoresis (12.5% SDS-PAGE). The proteins were transferred from the gel onto a nitrocellulose membrane (0.2 µm). The membranes were blocked in Roti-block solution (Carl Roth GmbH + Co., Karlsruhe, Germany) for 1 h. The membranes were stained with the polyclonal antibodies to DRP1 (Elabscience, Houston, TX, USA) at dilution 1:2000, Mfn2 (Elabscience, Houston, TX, USA) at dilution 1:2000, OPA1 (Cloud-Clone Corp., Katy, TX, USA) at dilution 1:1000, PHB2 (Cusabio, Houston, TX, USA) at dilution 1:1000, and PINK1 (Cusabio, Houston, TX, USA) dilution 1:500 overnight. The polyclonal anti-Tom20 antibodies (Cell Signaling, Danvers, MA, USA) at dilution 1:1000 were used to normalize the protein. Immunoreactivity was determined using secondary antibodies conjugated with horseradish peroxidase (Jackson Immuno Research, West Grove, PA, USA). Peroxidase activity was determined with an ECL (Bio-Rad, Hercules, CA, USA) using a ChemiDoc Touch imaging system (Bio-Rad, Hercules, CA, USA). Protein bands were quantified by densitometry (Image Lab software, Bio-Rad, Hercules, CA, USA) as the ratio of proteins to Tom20.
Roti block solution
Manufactured by Carl Roth
Sourced in Germany
Roti-Block solution is a blocking reagent used in Western blot analysis. It is designed to reduce non-specific binding of antibodies to the membrane, thereby improving the signal-to-noise ratio in the detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase conjugated secondary antibody, by Agilent Technologies (2 mentions)
Complete edta free, by Roche (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Anti tom20 antibody, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
0.2 m pvdf membrane, by Bio-Rad (1 mentions)
Odyssey fc, by LI COR (1 mentions)
Tween 20, by Merck Group (1 mentions)
Image studio software, by LI COR (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Anti tubulin clone aa13, by Merck Group (1 mentions)
Mouse anti flag clone m2, by Merck Group (1 mentions)
Mouse anti gapdh, by Merck Group (1 mentions)
Novex bis tris gradient gel, by Thermo Fisher Scientific (1 mentions)
Rabbit anti eaat2, by Abcam (1 mentions)
Nupage technical guide, by Thermo Fisher Scientific (1 mentions)
7 protocols using roti block solution
1
Mitochondrial Protein Analysis by Western Blot
2
Quantification of α-Synuclein in iPSC-Derived MSNs
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Conditioned medium from three independent differentiations of two control and two MSA-P (P1, P3) iPSC cell lines was collected, each at 40 and 70 ± 4 days of differentiation into MSNs. To remove cell debris, samples were spun at 1000× g for 10 min. Using vacuum dot blot equipment, the proteins were transferred on 0.2 µm PVDF membranes (Bio-Rad Laboratories). After blocking the membranes for 2 h at room temperature with 30% RotiBlock solution (Carl Roth, Karlsruhe, Germany), primary antibodies (Supplementary Table S1 ) were diluted accordingly and incubated with the membranes overnight at 4 °C. Membranes were washed (Tris-buffered saline (TBS) supplemented with 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA)), and the appropriate HRP-coupled secondary antibodies (Supplementary Table S2 ) were incubated for 1 h at room temperature. For visualization, the membranes were incubated in Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA), and the images were acquired using the Odyssey Fc (LICOR Biotechnology, Lincoln, NE) imaging system. Image Studio™ Software (version 5.2.5, LI-COR Biosciences GmbH, Bad Homburg vor der Höhe, Germany) was used for signal intensity annotation, and α-synuclein signals were normalized against GAPDH and β-actin signals.
3
Western Blot Analysis of Proteasome Subunits
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Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris pH 8, 1% (v/v) Triton X-100, 0.5% (v/v) sodiumdesoxycholate, 0.1% (w/v) SDS) including protease inhibitors (cOmplete EDTA-free, Roche) for 30 min on ice. Lysates were centrifuged at 14,000 rpm for 15 min and supernatants were boiled with SDS sample buffer for 5 min at 95 • C. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes (Whatman). After blocking in Roti-Block solution (Roth) for 1 h at room temperature, membranes were incubated with primary antibodies at 4 • C over night. Membranes were washed and incubated with appropriate peroxidase-conjugated secondary antibodies (Dako) for 2 h at room temperature. Membranes were washed again and proteins were visualized with enhanced chemiluminescence. Primary antibodies used were: anti-LMP7 and anti-LMP2 (Kremer et al., 2010) , anti-5 (D1H6B, Cell Signaling Technology), anti-1 (clone E1K90, Cell Signaling Technology), anti-␣1 (clone IB5, K. Scherrer, Paris, France), anti-IB␣ (clone L35A5, Cell Signaling Technology), anti-␣-tubulin (clone AA13, Sigma).
4
Immunoblot Analysis of Protein Lysates
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Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris pH 8, 1% (v/v) Triton X-100, 0.5% (v/v) sodiumdesoxycholate, 0.1% (w/v) SDS) including protease inhibitors (cOmplete EDTA-free, Roche) for 30 min on ice. Lysates were centrifuged at 20,000 × g and 4 • C for 15 min. Supernatants were mixed with SDS sample buffer and boiled for 5 min at 95 • C. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membrane (Whatman). Membranes were blocked for 1 h in Roti-Block solution (Roth) followed by overnight incubation in primary antibodies at 4 • C. Membranes were washed and incubated for 2 h with appropriate peroxidaseconjugated secondary antibodies (Dako). Membranes were washed and proteins were visualized with enhanced chemiluminescence. Primary antibodies used: Rabbit anti-BAG6 (kind gift from R. S. Hegde, Cambridge, UK), mouse anti-␣-tubulin (clone AA13, Sigma) and mouse anti-FLAG (clone M2, Sigma).
5
Western Blot Analysis of EAAT2 and GAPDH
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Samples were prepared according to the NuPAGE Technical Guide of Invitrogen. Briefly, after denaturation in NuPAGE LDS sample buffer with DTT 50 mM for 10 min at 70°C the samples and markers were run on a Novex bis-tris gradient gel (4–12%, Thermo Fischer Scientific) using NuPAGE MOPS SDS running buffer and subsequently blotted on a Novex 0.45 μm nitrocellulose membrane (LC2001, Thermo Fischer Scientific). The membranes were washed and blocked in ReadyTector solution A (CANDOR Bioscience GmbH, Wangen im Allgäu), and the primary antibodies rabbit anti-EAAT2 1:2000 (Abcam, Cambridge, United Kingdom) and mouse anti-GAPDH 1:1000 (Sigma-Aldrich) were directly applied for 1 h at room temperature in solution B that also contained horseradish peroxidase (HRP) coupled to the secondary antibodies against rabbit or mouse, respectively. To detect mYFP, membranes were blocked for 30 min in 1× Roti-block solution (Carl Roth GmbH, Karlsruhe) before applying rabbit anti-GFP 1:1000 (ChromoTek GmbH, Martinsried) over night at 4°C in the same solution. Then the HRP-coupled secondary antibodies (Dianova GmbH, Hamburg) were applied at 1:2500 for 3 h at 4°C. Proteins were detected using respective kits from Biozym Scientific GmbH (Hessisch Oldendorf). Proteins were detected by chemiluminescence and submitted to image analysis with ImageJ.
6
Western Blot Analysis of IMPDH2
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For western blot analyses cells were collected 0, 6, 8, 12, 16, and 24 h after treatment, respectively. The cells were taken up in lysis buffer and stored at −80°C as described before (Grabiec et al., 2019 (link)). Protein concentrations were determined using the method of Bradford (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Barrington IL, United States) (Bradford, 1976 (link)) and 10 μg protein was loaded onto a 12.5% (w/v) sodium dodecyl sulfate–polyacrylamide gel. After gel electrophoresis, the proteins were blotted onto nitrocellulose membranes (Protran 0.45 μm, Amersham, Freiburg, Germany) and non-specific protein binding sites were blocked for 30 min with roti block solution (Carl Roth, Karlsruhe, Germany). The nitrocellulose membranes were incubated with the antibody against IMPDH2 (Table 1 ) [diluted 1:2000 in roti block solution containing 0.2% (v/v) Tween 20] until the next day at 4°C. After incubation with horseradish peroxidase conjugated antibodies (Table 1 ) the chemiluminescent (Luminata Forte, Merck) signal was detected with Fusion X (VWR, Radnor, PA, United States). For semi quantitative analysis, the relative signal intensities of the immunoreactive bands were determined and the IMPDH2 intensity was normalized to the ß-actin (Table 1 ) intensity. Fusion FX7 with FusionCapt Advance Solo software (VWR) was used for analysis.
7
Western Blot Analysis of Graft Tissue
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Protein extracts from graft tissue were prepared as described previously. 35 Protein concentrations were determined using Micro BCA protein Assay kit (Pierce Biotechnology, Rockford, IL). Equal amounts of protein (20 μg) were resolved on 15% reducing sodium dodecyl sulfate (SDS)-polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were blocked with 1× Roti-Block solution (Roth, Karlsruhe, Germany) diluted in 50 mM Tris-HCl, pH 7.6, 0.9% NaCl. Rabbit polyclonal antibodies (Abs) to active caspase-3 (Abcam, Cambridge, UK) were diluted 1:1 000 in 1x Roti-Block (Roth). To ensure equal protein loading, membranes were incubated with mouse monoclonal Abs to glyceraldehyde 3phosphate dehydrogenase (GAPDH) (Novus Biologicals, Littleton, CO), 1:20,000. Bound antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark), 1:5 000, using the chemiluminescent reagent Lumi-Light Western blotting substrate (Roche, Mannheim, Germany). Densitometric analyses were performed using a digital gel documentation system (Biozym, Hessisch Oldendorf, Germany). Data were divided by the values obtained for GAPDH on the same blot.
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