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Figure 3 | Journal of Neuroinflammation

Figure 3

From: The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro

Figure 3

Blocking SK3 channels reduces microglial iNOS and NO, and tyrosine nitration of target neurons. Microglia were grown on Transwell™ inserts, and treated with LPS (100 ng/ml, 24 h), with or without 100 nM apamin, 5 nM tamapin or 250 pM tamapin. Results are expressed as RFU/mg protein in each well (mean±SEM; # of cell cultures indicated on bars), normalized to the signal from untreated microglia. A. iNOS protein was monitored with a mouse monoclonal anti-iNOS antibody (1:200) and a Cy3-conjugated secondary antibody (1:500). LPS stimulation increased iNOS expression (p < 0.05), which was reduced by 100 nM apamin (*p < 0.05) or 5 nM tamapin (**p < 0.01), but not by 250 pM tamapin. B. Nitric oxide production was measured as nitrite accumulation, using the Griess assay. LPS increased NO production (p < 0.05), which was abrogated by 100 nM apamin (**p < 0.01) or 5 nM tamapin (**p < 0.01), but unaffected by 250 pM tamapin. C. Tyrosine nitrated proteins in the target neurons were monitored with a rabbit polyclonal antibody against nitrotyrosine residues (1:200) and Cy3-conjugated secondary antibody. LPS-stimulated microglia induced tyrosine nitration in neurons (p < 0.05), and this was abrogated by treating the microglia with 100 nM apamin (***p < 0.001) or 5 nM tamapin (**p < 0.01), but not by 250 pM tamapin.

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