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. 2003 Nov 3;100(23):13326–13331. doi: 10.1073/pnas.2235648100

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Copyright © 2003, The National Academy of Sciences

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Fig. 1. — PrP^C-induced ROS production through NADPH oxidase activity. Extracellular release of ROS was detected by using the fluorogenic reagent OxyBurst Green H₂HFF BSA. (a Inset) The PrP^C-mediated ROS production was followed during 60 min with 1C11 precursor cells stimulated by 10 μg·ml^-1 of PrP-targeted antibodies 1A8 (•) or Bar221 (▴). Background ROS production was also recorded with unstimulated cells (▪). (a) Antibodies SAF61, Bar221, 1A8, and SAF32 were used at 10 μg·ml^-1 to elicit ROS production with 1C11 precursor cells, either 1C11^5-HT serotonergic or 1C11^NE noradrenergic cells, and with GT1-7 hypothalamic cells. ROS release was completed within 45 min of stimulation. ROS levels at 45 min are expressed in fluorescence intensity per mg of protein. The basal level of ROS production was substracted to the shown signals of PrP^C-induced ROS release. (b) ROS release induced by Bar221-mediated PrP^C ligation was also monitored with the BW5147 lymphoid cell line. As a control, ROS production was followed in the BW5147-derived Thy1^-e mutant, deficient for GPI synthesis. (c) PrP^C stimulation induces NADPH oxidase p47^PHOX phosphorylation. After metabolic labeling with , 1C11 precursor, 1C11^5-HT and 1C11^NE cells were stimulated with 1A8 antibody for 1 h. ³²P-labeled p47^PHOX (47 kDa) was immunoprecipitated from cytosolic extracts and detected as described in Materials and Methods. Control experiments (Ctrl) without antibody stimulation are also shown. (d) Involvement of NADPH oxidase in the PrP^C-induced ROS production was confirmed by using DPI, a specific inhibitor of NADPH oxidase activity. 1C11 precursor cells were preincubated with DPI concentrations ranging from 1 to 100μM and exposed to PrP antibodies in the presence of DPI. ROS release was followed over a period of 45 min. Shown are the mean ROS release velocities deduced from these experiments. Values of fluorescence intensity per mg of protein per time unit are normalized with respect to the value in the absence of DPI (100%). Data shown are representative of a set of three independent experiments.

Inline graphic — PrP^C-induced ROS production through NADPH oxidase activity. Extracellular release of ROS was detected by using the fluorogenic reagent OxyBurst Green H₂HFF BSA. (a Inset) The PrP^C-mediated ROS production was followed during 60 min with 1C11 precursor cells stimulated by 10 μg·ml^-1 of PrP-targeted antibodies 1A8 (•) or Bar221 (▴). Background ROS production was also recorded with unstimulated cells (▪). (a) Antibodies SAF61, Bar221, 1A8, and SAF32 were used at 10 μg·ml^-1 to elicit ROS production with 1C11 precursor cells, either 1C11^5-HT serotonergic or 1C11^NE noradrenergic cells, and with GT1-7 hypothalamic cells. ROS release was completed within 45 min of stimulation. ROS levels at 45 min are expressed in fluorescence intensity per mg of protein. The basal level of ROS production was substracted to the shown signals of PrP^C-induced ROS release. (b) ROS release induced by Bar221-mediated PrP^C ligation was also monitored with the BW5147 lymphoid cell line. As a control, ROS production was followed in the BW5147-derived Thy1^-e mutant, deficient for GPI synthesis. (c) PrP^C stimulation induces NADPH oxidase p47^PHOX phosphorylation. After metabolic labeling with , 1C11 precursor, 1C11^5-HT and 1C11^NE cells were stimulated with 1A8 antibody for 1 h. ³²P-labeled p47^PHOX (47 kDa) was immunoprecipitated from cytosolic extracts and detected as described in Materials and Methods. Control experiments (Ctrl) without antibody stimulation are also shown. (d) Involvement of NADPH oxidase in the PrP^C-induced ROS production was confirmed by using DPI, a specific inhibitor of NADPH oxidase activity. 1C11 precursor cells were preincubated with DPI concentrations ranging from 1 to 100μM and exposed to PrP antibodies in the presence of DPI. ROS release was followed over a period of 45 min. Shown are the mean ROS release velocities deduced from these experiments. Values of fluorescence intensity per mg of protein per time unit are normalized with respect to the value in the absence of DPI (100%). Data shown are representative of a set of three independent experiments.