Total protein was prepared from GFP-positive regions of ChR2-injected mPFC from air-exposed and CIE-exposed animals (N = 16). Ten micrograms of total protein was fractioned on 4–20% 18-well Criterion TGX Precast Midi Protein Gels (catalog #567-8094, Bio-Rad) and transferred to a nitrocellulose membrane with a Bio-Rad Trans Blot Turbo Transfer System and preassembled membrane stacks (catalog #1704270, Bio-Rad). Total lane protein transfer was detected using a Pierce Reversible Protein Stain Kit (catalog #PI24580, Thermo Fisher Scientific). GFP was targeted with chicken anti-GFP primary antibody (1 μg/ml; catalog #GFP-1020, Aves; RRID:AB_10000240 ), which was detected with peroxidase-labeled goat anti-chicken secondary antibody (1:5000; catalog #H-1004, Aves; RRID:AB_2313517 ) and SuperSignal West Dura Extended Duration Substrate Enhanced Chemiluminescence (catalog #34076, Thermo Fisher Scientific). Immunoreactive band intensity was quantified from digital images captured on a charge-coupled device camera and normalized to total lane protein using a Bio-Rad Chemi-Doc XRS Imaging System, and Image Lab Analysis software (Bio-Rad).
Pierce reversible protein stain kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce Reversible Protein Stain Kit is a laboratory product designed for the visualization and detection of proteins in polyacrylamide gels. It enables the staining and de-staining of proteins, allowing for their subsequent analysis or further processing.
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Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (4 mentions)
Igepal, by Merck Group (3 mentions)
Ha probe f 7 hrp, by Santa Cruz Biotechnology (3 mentions)
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Image lab analysis software, by Bio-Rad (1 mentions)
Criterion tgx precast midi protein gel, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Azure c300 gel dock, by Azure Biosystems (1 mentions)
Dry nitrocellulose membrane, by Bio-Rad (1 mentions)
Supersignal west pico kit, by Thermo Fisher Scientific (1 mentions)
Fast protease inhibitor, by Merck Group (1 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Rat monoclonal anti ha antibody 3f10, by Roche (1 mentions)
Ponceau s, by Merck Group (1 mentions)
Mini gel tank, by Thermo Fisher Scientific (1 mentions)
12 protocols using pierce reversible protein stain kit
1
Quantification of GFP Expression in ChR2-Injected mPFC
2
Western Blot Analysis of Protein Samples
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Protein samples were denatured in Laemmli buffer at 70°C for 10 min, then separated by SDS-PAGE. Separated samples were transferred to a nitrocellulose membrane (GE Healthcare, Pittsburgh, PA, USA). Total protein in each lane was detected using either Ponceau stain for immunoprecipitations, or the Pierce reversible protein stain kit (Thermo Fisher Scientific) for synaptoneurosome Western blots, then imaged using an Azure c300 gel dock (Azure Biosystems, Dublin, CA, USA). Membranes were blocked in 5% milk + 1× tris-buffered saline (TBS; 10×: 152.3 mM Tris-HCl, 46.2 mM Tris base, 1.5 M NaCl, pH 7.6) for 30 min at RT, then incubated in primary antibody in 1× TBS for either 1 h at RT or overnight at 4°C. Membranes were washed 3 × 10 min in 1× TBS, then incubated in an HRP-conjugated secondary antibody in block for 1 h at RT. After 3 × 10 min in 1× TBS, a chemiluminescent kit (Bio-Rad, Hercules, CA, USA) was used to detect the protein bands, and the membranes were imaged on a c300 gel dock.
3
Detecting Extracellular CXCL13 in RPE Cells
4
Protein Extraction and Immunoblotting from N. benthamiana Leaves
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Six N. benthamiana leaf discs (8 mm diameter) taken 5 days post agroinfiltration were homogenised in extraction buffer [10% glycerol, 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% (w/v) PVPP, 10 mM DTT, 1x protease inhibitor cocktail (SIGMA), 0.2% IGEPAL CA-630 (SIGMA)]. The supernatant obtained after centrifugation at 12,000 x g for 10 min 4 °C was used for SDS-PAGE. Immunoblotting was performed with rat monoclonal anti-HA antibody (3F10, Roche) or mouse monoclonal anti-GFP antibody conjugated to HRP (B-2, Santa Cruz Biotech) in a 1:5000 dilution. Equal loading was validated by staining the PVDF membranes with Pierce Reversible Protein Stain Kit (#24585, Thermo Fisher) or using Ponceau S (SIGMA).
5
SDS-PAGE and Western Blot Analysis
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Protein samples were mixed with 10× NuPAGE Sample Reducing Agent (ThermoFisher Scientific) and 4× NuPAGE LDS Sample Buffer (ThermoFisher Scientific), denatured at 95 °C for 5 min and applied to a polyacrylamide gel (Novex NuPAGE 4–12% Bis-Tris protein-gel, 1.0 mm, 10-well; ThermoFisher Scientific) using a mini gel tank (ThermoFisher Scientific). SDS-PAGE was performed at 200 V for 60 min with MOPS running buffer (ThermoFisher Scientific). Then the proteins were transferred onto a 0.45 µm pore size nitrocellulose membrane (ThermoFisher Scientific) at 30 V for 75 min via semidry blotting (Novex Semi-Dry Blotter, ThermoFisher Scientific) in NuPAGE transfer buffer. Pierce Reversible Protein Stain Kit (ThermoFisher Scientific) was used to stain whole blotted protein before detection of Strep-tagged proteins, which was carried out using Strep-MAB-Classic (HRP conjugate, IBA GmbH) based chemiluminescence detection and Clarity Western ECL substrate (Bio-Rad Laboratories, Munich, Germany) according to the manufacturers’ protocols. A ChemiDoc MP imaging system (Bio-Rad Laboratories) and image Lab 5.2 software (Bio-Rad Laboratories) were used for documentation.
6
Western Blot Analysis of Protein Expression
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Samples were mixed with 4x laemlli buffer and incubated at 95°C for 5 mins. Proteins were loaded and separated by 10% or 12% SDS-PAGE gel, followed by wet transfer to a nitrocellulose membrane (GE Healthcare, Chicago, IL). Total protein was stained and destained by Pierce Reversible Protein Stain Kit (Thermo Fisher Scientific, Waltham, MA). The membrane was blocked by 5% milk in TBS for 1h at room temperature. Primary antibodies were diluted in 1% milk in TBS and incubated with the membrane at 4°C overnight. Primary antibodies include: anti-myc (05–724, Sigma-Aldrich, St. Louis, MO; 1:1000), anti-PNMA2 (16445-1-AP, Proteintech, Rosemont, Illinois; 1:1000), anti-Alix (customized antibody from Wesley Sundquist’s lab; 1:500), mice sera (1:1000). The membrane was washed by TBS and then incubated with secondary antibodies (anti-mouse IgG, anti-rat IgG, anti-human IgG, Jackson Laboratory, Bar Harbor, ME; 1:5000) diluted by 1% milk in TBS for 1h at room temperature. Afterwards, the membrane was washed with TBS for three times. Bound antibodies were detected by Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA) and imaged using an Amersham ImageQuant™ 800 Western blot imaging systems (Cytiva, Marlborough, MA). Images were analyzed and quantified using ImageJ.
7
Quantification of GM-CSF in Mouse Milk
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Milk was collected with a pipette from narcotized female mice at day 10 of lactation after oxytocin administration and stored at − 80 °C. For ELISA analysis, milk was diluted 1–6 million times with water and assayed according to the manufacturer’s recommendation (Human GM-CSF Quantikine ELISA Kit, DGM00, R&D Systems). Blood was collected from the euthanized mice and the serum was diluted × 1000 times for the assay. Measurements were taken at 490 nm with BioTek Epoch Spectrophotometer.
The protein concentrations in milk were quantified using Pierce BCA Protein Assay Kit (ThermoFisher). For Western blot, equal amounts (20 μg) of milk samples were separated on 15% SDS-PAGE, and then transferred onto Immun-Blot PVDF membrane (Bio-Rad). Membrane was blocked with 5% milk/TBST(20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 2 h and incubated with primary antibodies against hGMCSF 1:500 at 4 °C overnight (R&D, catalogue # AF-215-NA). Next day, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies 1:1000 (#93702, Cell Signaling) for 2 h at 25 °C. Detection was performed with SuperSignal West Pico PLUS Chemiluminescent Substrate (#34580, ThermoFisher) and Chemidoc XRS Imaging system (Bio-Rad). Reversible staining of the PVDF membrane (Fig.3 A) was performed with Pierce Reversible Protein Stain Kit (#24585, ThermoFisher).
The protein concentrations in milk were quantified using Pierce BCA Protein Assay Kit (ThermoFisher). For Western blot, equal amounts (20 μg) of milk samples were separated on 15% SDS-PAGE, and then transferred onto Immun-Blot PVDF membrane (Bio-Rad). Membrane was blocked with 5% milk/TBST(20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 2 h and incubated with primary antibodies against hGMCSF 1:500 at 4 °C overnight (R&D, catalogue # AF-215-NA). Next day, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies 1:1000 (#93702, Cell Signaling) for 2 h at 25 °C. Detection was performed with SuperSignal West Pico PLUS Chemiluminescent Substrate (#34580, ThermoFisher) and Chemidoc XRS Imaging system (Bio-Rad). Reversible staining of the PVDF membrane (Fig.
8
Western Blot Analysis of Heart Proteins
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Heart lysates were prepared in RIPA buffer supplemented with Halt protease inhibitor cocktail as described (Wingard et al., 2021 (link)). Equal amounts of proteins (50 μg) were resolved by SDS‐PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat dry milk for 1 hour and incubated overnight with primary antibodies against MMP‐9 (1:1000, Cat# AB19016; Millipore), MMP‐2 (1:500, Cat# MAB3308; Millipore), Bax (1:1000, Cat# SC7480; Santa Cruz), PARP‐1 (1:1000, Cat# 9542; Cell Signaling), p‐Akt (ser473; 1:1000, Cat# 9271S; Cell Signaling), and p‐mTOR (ser‐2448; 1:1000, Cat# 5536S; Cell Signaling). The immune complexes were detected using appropriate secondary antibodies and chemiluminescent reagents (Wingard et al., 2021 (link)). Because assessment of loading using a total protein stain is considered as a better approach (Brooks & Lindsey, 2018 (link)); western blot data were normalized using Pierce reversible protein stain kit (Thermo Fisher). The portion of the membrane used for normalization is shown in Figures 6 , 7 , 8 .
9
Protein Extraction and Analysis from N. benthamiana
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Protein samples were prepared from six discs (8 mm diameter) cut out of N. benthamiana leaves at 2 days after agroinfiltration and were homogenised in extraction buffer [10% (v/v) glycerol, 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 2% (w/v) PVPP, 10 mM DTT, 1x protease inhibitor cocktail (SIGMA), 0.5% (v/v) IGEPAL (SIGMA)]. The supernatant obtained after centrifugation at 12,000 xg for 10 min was used for SDS-PAGE. Immunoblotting was performed with HA-probe (F-7) HRP (Santa Cruz Biotech) in a 1:5,000 dilution. Equal loading was checked by taking images of the stained PVDF membranes with Pierce Reversible Protein Stain Kit (#24585, Thermo Fisher Scientific).
10
Protein Extraction and Detection in N. benthamiana
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Protein samples were prepared from six discs (8 mm diameter) cut out of N. benthamiana leaves at 1 day after agroinfiltration and were homogenised in extraction buffer [10% glycerol, 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 2% (w/v) PVPP, 10 mM DTT, 1x protease inhibitor cocktail (SIGMA), 0.2% IGEPAL (SIGMA)]. The supernatant obtained after centrifugation at 12,000 xg for 10 min was used for SDS-PAGE. Immunoblotting was performed with HA-probe (F-7) HRP (Santa Cruz Biotech) or anti-GFP antibody (ab290, abcam) in a 1:5000 dilution. Equal loading was checked by taking images of the stained PVDF membranes with Pierce Reversible Protein Stain Kit (#24585, Thermo Fisher).
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