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. 2021 Jul 10;297(2):100946. doi: 10.1016/j.jbc.2021.100946

Figure 3.

Figure 3

Nitrosative stress induced by LPS in vivo leads to PKG-Iα tyrosine nitration.A, peroxynitrite levels, estimated using DHR oxidation, were significantly increased in LPS-treated mice. Specificity of signal was demonstrated by MnTMPyP pretreatment attenuating DHR123 oxidation. B, immunoprecipitation analysis using an antibody specific to nitrotyrosine (3-NT) demonstrated an increase in the tyrosine nitration of PKG-Iα immunoprecipitated from LPS-exposed tissues. MnTMPyP pretreatment prevented the nitration of PKG-Iα. IgG (HC) is the immunoglobulin heavy chain. C, immunoblotting using an antibody specific to nitro-Y247 in PKG-Iα confirmed the increase in PKG-Iα nitration in HEK293T cells transfected with a PKG-Iα expression plasmid and treated with 3-morpholinosydnonimine. D, an increase in PKG-Iα nitration in mouse lungs exposed to LPS was also shown to occur at Y247. MnTMPyP reduced Y247 nitration in PKG-Iα in response to LPS. Data represent mean ± SEM, n = 3 to 5. ∗p < 0.05 versus control lungs; †p < 0.05 versus LPS alone. DHR, dihydrorhodamine; HEK293T, human embryonic kidney 293T cells; LPS, lipopolysaccharide; MnTMPyP, manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin; PKG-Iα, protein kinase G-Iα; Y247, tyrosine 247.