Lysates generated from BMDMs, were separated by 15 % SDS-PAGE and transferred onto nitrocellulose PVDF membranes (Millipore). Blots were immersed in 5 % milk powder in TBS/tween and incubated overnight at 4 °C with a rat monoclonal antibody for IL-1β (RnD Systems AF-501). Membranes were then washed and incubated with rabbit anti-rat secondary antibody (Abcam). Proteins were imaged using ECL Plus chemi luminescent substrate (ThermoFisher Scientific) and exposed using the ChemiDoc XRS imaging system (BioRad Laboratories).
Ecl plus chemiluminescent substrate
Manufactured by Thermo Fisher Scientific
Sourced in France
ECL Plus chemiluminescent substrate is a detection reagent used in western blotting applications. It generates a luminescent signal upon reaction with the horseradish peroxidase (HRP) enzyme, allowing for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Nitrocellulose pvdf membranes, by Merck Group (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Hrp conjugated goat anti rabbit antibody, by Cell Signaling Technology (1 mentions)
Mouse monoclonal anti acetyl lysine antibody, by Cell Signaling Technology (1 mentions)
Mouse anti strep tag 2 monoclonal antibody, by Abbkine (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Mouse anti his tag antibody, by Proteintech (1 mentions)
Cl xposure film, by Thermo Fisher Scientific (1 mentions)
Anti a2ar, by Merck Group (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
β actin a1978, by Merck Group (1 mentions)
Trans blot sd, by Bio-Rad (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Twist 2c1a, by Santa Cruz Biotechnology (1 mentions)
Blue devil film, by Genesee Scientific (1 mentions)
10 protocols using ecl plus chemiluminescent substrate
1
Immunoblotting Protocol for IL-1β Detection
2
Western Blot Analysis of Protein Samples
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Loading buffer with additional β-Me (2%) to denature the protein sample, protein samples in SDS-PAGE sample buffer were boiled for 8 min, resolved on an SDS-PAGE gel, and transferred to PVDF membrane (Thermo Fisher Scientific). The membrane was blocked in 5% skim milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) at room temperature for 1 hour. Then the membrane was incubated with mouse anti-His tag antibody (1:10000, Proteintech, Cat. No. HRP-66005), mouse anti-vinculin monoclonal antibody (1:2000, Cat. No. 66305-1-Ig), mouse monoclonal anti-acetyl lysine antibody (1:1000, Cell Signaling Technology, Cat. No. 9681), mouse anti-strep-tag II monoclonal antibody (1:2000, Abbkine, Cat. No. ABT2230), rabbit polyclonal FtsZ antibody (1:1000, CUSABIO, Cat. No. CSB-PA359270HA01EGX) in TBST at 4 °C overnight. The membrane was washed with TBST (4 × 5 min) before the addition of the secondary goat anti-mouse horseradish peroxidase conjugate (1:4000, Proteintech, Cat. No. SA00001-1) or HRP-conjugated goat anti-rabbit antibody (1:5000, Cell Signaling Technology, Cat. No. 7074). After 1 hour, the membrane was washed with TBST (4 × 5 min). Final detection of HRP activity was performed using ECL Plus chemiluminescent substrate (Thermo Fisher Scientific). The image was acquired with Image Quant LAS 4000 or ChemiScope 6300. Uncropped images can be found in the Source Data File.
3
Western Blot Analysis of Spinal Cord
4
Western Blot Analysis of TWIST Expression
5
Western Blotting Protocol for Otx2 and Smad Signaling
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Western blotting was performed as described previously (Yoshitomi et al., 2017 (link)). Briefly, cells were lysed in Laemmli buffer containing 5% 2-mercaptoethanol and were sonicated for 30 s and then heated at 98°C for 5 min. The resulting protein samples were subjected to SDS-PAGE using 4%–12% polyacrylamide gels (Wako, Osaka, Japan). After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Invitrogen) and were blocked with 2% BSA in Tris-buffered saline. Membranes were then incubated with Anti-Otx2 antibody (cat. no. AF1979; R&D Systems, Minneapolis, MN), anti-Phospho-Smad2 (Ser465/467) (Clone no. 138D4, Cat. no. 3108, 1:1000, Cell Signaling Technology, Danvers, MA), anti-Phospho-Smad3 (Ser423/425) (Clone no. C25A9, Cat. no. 9520, 1:1000, Cell Signaling Technology), anti-Smad2/3 (Clone no. D7G7, Cat. no. 8685, 1:1000, Cell Signaling Technology), and anti-β-actin (Cat. no. A2228, 1:4000; Sigma-Aldrich) primary antibodies, followed by appropriate HRP conjugated secondary antibodies (Amasham GE, MA). Protein signals were then detected using ECL Plus Chemiluminescent Substrate (Thermo Fisher Scientific, Rockford, IL) with a Vilber-Lourmat FUSION FX7 imaging system (Vilber, Collégien, France).
6
Quantitative Protein Biotinylation Assay
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Supernatants from BMDMs that had been treated with LLOME for the indicated time-points were diluted in MES reaction buffer and incubated at 37 °C for 15 mins with 10 μM biotin-PEG-LVG-DMK. The reaction was terminated by addition of 5X Laemmli buffer and denaturation at 95 °C. Samples were resolved by 15 % SDS-PAGE and transferred onto nitrocellulose PVDF membranes (Millipore), blots were subsequently blocked in 5 % BSA in TBS/tween overnight at 4 °C. The membrane was then incubated with Streptavidin-horse radish peroxidase (Cell Signaling) diluted TBS/Tween, with 5 % BSA for 0.5 h at room temperature. Proteins were imaged using ECL Plus chemiluminescent substrate (ThermoFisher Scientific) and exposed using the ChemiDoc XRS imaging system (BioRad Laboratories).
7
Western Blot Quantification Protocol
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Protein quantity was determined for SDS boiled lysates using the EZQ Protein Quantitation Kit (Life Technologies) following the manufacturer’s instructions. For each sample a total of 10 μg of protein lysate was subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% skim milk powder for 1 h. Membranes were washed three times (10 min each) with Tris-buffered saline/Tween 20 (TBST), and then incubated for 1 h with primary antibody diluted in TBST. The following primary rabbit antibodies were used: CD40 (1:5,000, Thermo Fisher, Waltham, USA), A20 (1:1,000, Cell Signaling, Danvers, USA) and β2-tubulin (1:2,000, Cell Signaling). Membranes were washed three times with TBST and then incubated for 1 h with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody diluted in TBST. Membranes were finally washed twice with TBST, once with TBS and developed with ECL-Plus chemiluminescent substrate (Thermo Fisher) as per the manufacturer’s instructions. Membranes were scanned at 473 nm on a Typhon FLA9000 gel imaging scanner (GE Healthcare, Little Chalfont, UK).
8
Western Blot Analysis of Exosomal Proteins
9
Exosome and Tissue Protein Analysis
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Isolated APPNL-F and WT exosome samples were lysed with 5× RIPA buffer with Triton X-100 (AAJ62885AD, Fisher) and 25× P8340 (protease inhibitor cocktail) to a final concentration of 1× RIPA +1% P8340. Total protein amount for exosome and cortical homogenate samples was determined using a DC™ Protein Assay Kit II (Bio-Rad) per manufacturer’s instructions. For exosome samples, 30 μL of the final lysate was loaded on 4–15% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad). Tissue homogenates were diluted for a final loading protein content of 30 μg (20 μL loaded per lane). Separated proteins were then transferred onto a PVDF membrane (Bio-Rad) with the Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was blocked with 5% fat-free skim milk in TBST (Tris-buffered saline +0.05% Tween-20) or Super-Block™ T20 (TBS) Blocking Buffer (Thermo Scientific) then incubated with primary antibody overnight at 4 °C (Alix [clone 1A12], 1:500, Santa Cruz; human APP [clone 6E10], 1:1000, BioLegend). Secondary antibodies, including ECL anti-mouse IgG (1:10000, GE Healthcare NA931V) are diluted with Super Blocking Buffer. Bands were visualized on Chemidoc MP imaging system (Bio-Rad) with ECL Plus chemiluminescent substrate (Thermo Fisher Scientific) or Clarity Max Western ECL Substrate (Bio-Rad).
10
Western Blot Analysis of EPAC2 Protein
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Cells were suspended in Laemmli-sample buffer containing 0.2 µM DTT and shredded using a QIA shredder (Qiagen) and heated at 98 °C for 5 min. The resulting protein samples were applied to SDS-PAGE using 7.5% polyacrylamide gel (ATTO). After electrophoresis, proteins were transferred onto PVDF membranes (ATTO) and blocked with 5% skim milk in 0.1% Tween 20/PBS (TPBS). Anti-EPAC2 (5B1, Cell Signaling Technology) or anti-β-actin (Sigma-Aldrich) antibodies in 1% BSA/TPBS were used as primary antibodies, and anti-mouse IgG-HRP (Cell Signaling Technology) or anti-rabbit IgG-HRP (GE Healthcare) in 1% BSA/TPBS were used as secondary antibodies. Protein signals were detected using an ECL Plus Chemiluminescent Substrate (Thermo Fisher Scientific) using Molecular Dynamics Typhoon 9400 Imager.
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