A human phosphokinase array kit (ARY003B; R&D Systems) was used to investigate the effects of incubating IL-1β–stimulated patient-derived tendon cells in LXB4 or RvE1 on protein kinase signaling pathways (n = 3 donors). Experimental protocols were performed according to the manufacturer's instructions on protein lysates harvested after 24 hours of incubation in LXB4 or RvE1. Images were captured using a chemiluminescence documentation system (UVITEC, Cambridge, UK), and densitometry analysis of proteins of interest was performed using ImageJ software version 1.47v (ImageJ bundled with Java 1.8.0_172, NIH, Bethesda, MD; https://imagej.nih.gov/ij ).
Chemiluminescence documentation system
Manufactured by Uvitec
Sourced in United Kingdom
The Chemiluminescence Documentation System is a specialized lab equipment designed to detect and document chemiluminescence reactions. It provides a precise and reliable method for capturing and analyzing the light emission generated during chemical reactions.
Automatically generated - may contain errors
4 protocols using chemiluminescence documentation system
1
Phosphokinase Profiling of Tendon Cells
2
Phosphokinase Signaling Modulation by Lipid Mediators
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A Human Phosphokinase Array Kit (ARY003B; R&D Systems, Minneapolis, MN, USA) was used to investigate the effects of incubating IL-1β–treated AT cells in 15-epi-LXA4 or MaR1 on protein phosphokinase signaling pathways. Experimental protocols were performed according to the manufacturer’s instructions on protein lysates harvested after 24 h of incubation in either 15-epi-LXA4 or MaR1. Images were captured using a chemiluminescence documentation system (Uvitec, Cambridge, United Kingdom), and densitometry analysis of proteins of interest was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Protein Nitrosylation and Nitration Analysis
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Total cell lysates (50 μg/mL) were subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA, United States). Blots were probed using specific monoclonal antibodies against Nitroso-Cys (Ag Scientific) and Nitro-Tyr (Cell Signaling) at a 1:1,000 dilution. After incubation with the appropriate HRP-conjugated secondary antibodies (at 1:2,000 dilution), blots were developed using the Super Signal system (Thermo-Pierce – Rockford, IL, United States). Image acquisition and densitometry were carried out using a chemiluminescence documentation system (UVITEC, Cambridge, United Kingdom).
4
Immunoblotting Analysis of Paracoccidioides Aspartyl Protease
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Western blot analysis was performed with sera from hyperimmune mice or patients with PCM. Purified rPbSap was electrophoresed on 12% SDS-PAGE gels and electroblotted to nylon membranes that were blocked with 5% (w/v) non-fat dried milk in 1x TBS-T for 1 h at room temperature with shaking. Aspartyl protease was detected using sera from mice containing anti-rPbSap polyclonal antibodies (produced in this work) or sera from PCM patients. After incubation with the appropriate HRP-conjugated secondary antibodies anti-mouse IgG (whole molecule) (at 1:2,000 dilutions, KPL), the blots were developed using the Super Signal system (Thermo-Pierce–Rockford, IL). Image acquisition and densitometry were performed using a chemiluminescence documentation system (UVITEC, Cambridge, UK). Negative controls included preimmune sera from mice or sera from healthy patients. Total protein extracts from yeast were obtained as described [22 ] and subjected to western blot analysis using the anti-rPbSap polyclonal antibody to detect PbSap in total protein extracts from Pb18 yeast cells.
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