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. 2012 Nov 15;17(10):1393–1406. doi: 10.1089/ars.2011.4485

FIG. 5.

FIG. 5.

Inactivation of PGHS-2 in a Cu/Zn-SOD overexpression model. To limit a potential involvement of peroxynitrite that originates from the interaction of •NO and •O2−, HEK 293T cells were transiently transfected to constitutively express PGHS-2 and Cu/Zn-SOD. The cells were additionally transfected with a construct coding for NOS-2 under the control of a doxycycline-dependent promoter. (A) HEK 293T cells were transfected with combinations of plasmids coding for PGHS-2, Cu/Zn-SOD, and NOS-2. Doxycycline (1 μg/ml) was added for 48 h and NO2−, as indicator for NOS-2 induction was detected in the supernatants. The respective cell pellets were homogenized, PGHS activity was detected by following the conversion of 14C-AA. The SOD activity was analyzed in parallel by the cytochrome c assay. PGHS-2 was then immunoprecipitated from the cell homogenate, subjected to SDS-gel electrophoresis, and probed with anti-PGHS-2 and anti-3-NT antibodies. (B) HEK 293T cells were transfected with plasmids coding for PGHS-2, Cu/Zn-SOD, and NOS-2. Doxycycline, required for the induction of NOS-2 was added for the time intervals as indicated and NO2− was detected in the supernatant as an indicator for the NOS-2 activity. The time-dependent accumulation of NO2− correlated with a decline in the PGHS activity and an increase in 3-NT staining of immunoprecipitated PGHS-2. Values are mean±SD (n=4). *p<0.05 versus AMT=0. •O2−, superoxide; SDS, sodium dodecyl sulfate.