Protein isolation from Psoriatic Arthritis synovial fibroblasts (PsAFLS) and synovial explants is described in online supplementary file 1. Protein (20–50 µg) was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (5% stacking, 10% resolving), gels were then transferred onto nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK) prior to 1 h blocking in wash buffer containing 5% non-fat milk. Membranes were incubated with rabbit polyclonal anti-pSTAT3 (Cell-Signaling Technology, UK), total-signal transducer and activator of transcription (tSTAT)3, pSTAT1, tSTAT1, pSTAT2, suppressor of cytokine signaling-3 (SOCS3), protein inhibitor of activated Stat3 (PIAS3; Cell Signaling Technology) and nuclear factor kappa B cells (NFκBp65) (Millipore, California, USA) diluted in 5% non-fat milk containing 0.1% Tween 20 at 4°C overnight. β-Actin (Sigma-Aldrich) was used as a loading control. Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 3 h at RT. Signal was detected using SuperSignal West-Pico Chemiluminescent Substrate (Amersham Biosciences, UK) and quantified using EDAS-120 system (Kodak, Rochester, New York, USA).
Edas 120 system
Manufactured by Kodak
Sourced in United Kingdom, United States
The EDAS-120 system is a laboratory equipment designed for image analysis and data processing. It is a comprehensive solution that provides advanced image capture, enhancement, and analysis capabilities. The EDAS-120 system offers high-resolution image acquisition, intuitive software interfaces, and powerful data processing tools, enabling users to perform a range of analytical tasks in various research and industrial applications.
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Lab products found in correlation
Supersignal west pico chemiluminescent substrate, by Cytiva (2 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Nf κb p65, by Merck Group (1 mentions)
Anti p stat3, by Cell Signaling Technology (1 mentions)
Pias3, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Anti actin, by Merck Group (1 mentions)
2 protocols using edas 120 system
1
Profiling Synovial Fibroblast Signaling in Psoriatic Arthritis
2
Western Blot Analysis of Protein Expression
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Protein expression was evaluated by western blot, using standard sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) techniques as previously described (8 (link)). Briefly, harvested cells were lysed in 5X Laemmli sample buffer (5 mmol/L Na3VO4, 2.5 mmol/L p NPP, 10 mmol/L NaF, 5 mmol/L EDTA, 5 mmol/L EGTA, 20 μg/L leupeptin, 20 μg/L pepstatin and 20 μg/L aprotinin) and incubated for 3 min at 70°C and stored at −80°C until use. Lysates were sonicated and subjected to 10% SDS-PAGE. The separated proteins were transferred to nitrocellulose and blocked in 5% milk in Tris-buffered saline containing Tween 20 (TBS-T) for 1 h. The membranes were then probed overnight with primary antibodies-anti-gp91/NOX2 (Santa Cruz 1:5,000), anti-NOX5 (Abcam 1:1,000), and anti-actin (Sigma 1:10,000). Membranes were washed and incubated with HRP-labeled specific antibody. The signal was detected using SuperSignal® West Pico Chemiluminescent Substrate (Amersham Biosciences) and density of the bands was analyzed using EDAS 120 system from Kodak (Kodak, Rochester, NY, USA).
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