To prevent light-induced degradation of NO donors or protein nitrosothiols, all reagents/samples were protected from light by covering tubes and containers with foil during in vitro SNO reaction. Endotoxin-free recombinant HMGB1 protein (2.5 μg/mL in sterile normal saline; Biolegend; Cat# 764,006) or saline was incubated with NO donor GSNO (50 μM) or saline in the dark for 1 h at 37°C. Then 50 μL of the reaction mixture was added to midbrain neuron-glia cultures containing 450 μL of the treatment medium. Thus, after possible decomposition of GSNO during 1-h in vitro incubation at 37°C, the medium contained less than 5 μM GSNO with or without 250 ng/mL SNO-HMGB1. The four treatment groups were called “Saline”, “HMGB1”that contained unmodified HMGB1, “incubated GSNO” or “SNO-HMGB1”. At 7 days after the treatment, neurodegeneration was evaluated by Immunocytochemistry, cell count and Immunoblotting.
Hmgb1 protein
Manufactured by BioLegend
HMGB1 protein is a highly conserved nuclear protein that functions as a DNA-binding protein and plays a role in various cellular processes. It is involved in the regulation of gene expression, DNA repair, and the maintenance of chromatin structure. The HMGB1 protein is found in the nucleus of eukaryotic cells and can be released into the extracellular space, where it can act as a signaling molecule and play a role in inflammatory responses.
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Lab products found in correlation
2 protocols using hmgb1 protein
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Nitrosation of HMGB1 and neurodegeneration
2
Nitrosation of HMGB1 and neurodegeneration
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To prevent light-induced degradation of NO donors or protein nitrosothiols, all reagents/samples were protected from light by covering tubes and containers with foil during in vitro SNO reaction. Endotoxin-free recombinant HMGB1 protein (2.5 μg/mL in sterile normal saline; Biolegend; Cat# 764,006) or saline was incubated with NO donor GSNO (50 μM) or saline in the dark for 1 h at 37°C. Then 50 μL of the reaction mixture was added to midbrain neuron-glia cultures containing 450 μL of the treatment medium. Thus, after possible decomposition of GSNO during 1-h in vitro incubation at 37°C, the medium contained less than 5 μM GSNO with or without 250 ng/mL SNO-HMGB1. The four treatment groups were called “Saline”, “HMGB1”that contained unmodified HMGB1, “incubated GSNO” or “SNO-HMGB1”. At 7 days after the treatment, neurodegeneration was evaluated by Immunocytochemistry, cell count and Immunoblotting.
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