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Supersignal west pico plus chemiluminescence substrate kit

Manufactured by Thermo Fisher Scientific

The SuperSignal West Pico PLUS Chemiluminescence Substrate Kit is a laboratory reagent designed to enable chemiluminescence detection of proteins in Western blot applications. The kit provides a chemiluminescent substrate solution that can be used with horseradish peroxidase-conjugated detection systems.

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4 protocols using supersignal west pico plus chemiluminescence substrate kit

1

Western Blot Profiling of CD8+ T Cells

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CD8+ T cells from healthy donors were pelleted via centrifugation at 3,000 rpm for 15 minutes and then lysed in NP-40 buffer (20 mmol/L Tris-CL, pH 8.0, 137 mmol/L NaCl, 10% glycerol, 1% NP-40, 2 mmol/L EDTA) and concentrations measured via protein assay using Bio-Rad reagent (catalog no. 500-0006). Once diluted to equal concentrations, samples were combined with Laemmli sample buffer (Bio-Rad, catalog no. 1610747) and run using a Mini-PROTEAN Electrophoresis and Transfer system (Bio-Rad, catalog no. 1658004). Membranes were incubated with primary antibody overnight at 4°C (antibodies used were specific for NKG7, β-actin, ETS-1, or GAPDH; details listed in Supplementary Table S3). Secondary antibodies were added the next morning for 1 hour [horseradish peroxidase (HRP)–conjugated anti-rabbit and anti-mouse; details in Supplementary Table S3]. Signal was visualized using SuperSignal West Pico PLUS Chemiluminescence Substrate Kit (catalog no. 34577, Thermo Fisher Scientific) on a Syngene G:Box Chemi XX6 system running GeneSys software (V1.6.1.0; RRID:SCR_015770). Images were formatted for figures using Microsoft PowerPoint (Microsoft Office 2016). Full images are included in the Supplementary Data File S1.
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2

EV Protein Immunoblotting Procedure

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One-third of EV proteins were separated on a 4–15% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The membrane was incubated with 5% milk for 1 h at room temperature (RT) and then incubated with anti-CD9 antibody (1:1000) overnight at 4 °C followed by incubation with HRP conjugated secondary antibody (1:5000) for 1 h at RT. The membrane was washed with PBST (1× PBS, 0.1% Tween) for 10 min three times between each step and finally visualized using a SuperSignal West Pico Plus chemiluminescence substrate kit (Thermo). The membrane was incubated with the chemiluminescence substrate for 5 min prior to exposure on an imaging system (Bio-Rad).
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3

Western Blot Analysis of Fluorescent Proteins

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All the samples obtained during the FLAG Pull-down assay were separated by SDS-PAGE using 12% gels and transferred to the Whatman Protran nitrocellulose transfer membrane (Protran BA85, Schleicher & Schuell Pure, Sigma-Aldrich) in the semi-dry system at 10 V for 40 min in Towbin buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3). The membranes were blocked at room temperature with 2% milk powder (Milchpulver, blotting grade, Roth) in the PBS buffer and incubated for 1 h at room temperature. Next, the membrane was incubated overnight at 4 °C with the specific primary anti-GFP polyclonal antibodies (Sigma-Aldrich) (diluted 1:300 with milk buffer), which cross-react with CFP and YFP. After washing (PBS supplemented with 0.02% Tween, 3 × 10 min), the membrane was incubated for 2 h with secondary goat anti-mouse antibodies coupled to horseradish peroxidase (Vector Laboratories, dilution 1:10000 with milk buffer). Specific signals were detected using the SuperSignal™ West Pico PLUS Substrate Chemiluminescence kit (Thermo Scientific™) according to the manufacturer’s manual. Finally, the membranes were exposed to Kodak BioLight film.
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4

Western Blotting of Fluorescent Fusion Proteins

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All the samples obtained during the FLAG Pull-down assay were separated by SDS-PAGE using 12% gels and transferred to the Whatman Protran nitrocellulose transfer membrane (Protran BA85, Schleicher & Schuell Pure, Sigma-Aldrich) in the semi-dry system at 10 V for 40 min in Towbin buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3). The membranes were blocked at room temperature with 2% milk powder (Milchpulver, blotting grade, Roth) in the PBS buffer and incubated for 1 h at room temperature. Next, the membrane was incubated overnight at 4 °C with the specific primary anti-GFP polyclonal antibodies (Sigma-Aldrich) (diluted 1:300 with milk buffer), which cross-react with CFP and YFP. After washing (PBS supplemented with 0.02% Tween, 3x10 min), the membrane was incubated for 2 h with secondary goat anti-mouse antibodies coupled to horseradish peroxidase (Vector Laboratories, dilution 1:10 000 with milk buffer). Specific signals were detected using the SuperSignal™ West Pico PLUS Substrate Chemiluminescence kit (Thermo Scientific™) according to the manufacturer's manual. Finally, the membranes were exposed to Kodak BioLight film.
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