Figure 2

A: Western blots detecting iNOS in total protein extracts from PC12 cells treated for 24 hr with increasing concentrations of NGF in the presence or absence of 10 ng/ml TNFα (Top) or treated with increasing concentrations of TNFα in the presence or absence of 25 ng/ml NGF (Bottom). Positive control (Pos) is 4 μg of total protein extracts from mouse macrophages. Results shown are representative of 2 replicate experiments. B: Western blot analysis detecting iNOS in total protein extracts from PC12 cells simultaneously pre-treated with 10 ng/ml NGF and 10 ng/ml TNFα. At 24 hr treatment was withdrawn and the presence of iNOS was determined 24, 48, and 72 hr thereafter. Results shown are representative of 3 replicate experiments. C: Western blot analysis (Top) and RT-PCR (Bottom) detecting iNOS protein and mRNA in total protein extracts and total RNA from PC12 cells simultaneously pre-treated for 24 hr with 10 ng/ml NGF and 10 ng/ml TNFα (Both). After 24 hr, treatment was withdrawn and replaced with either NGF or TNFα alone or with both and iNOS expression determined 24 hr thereafter. Results shown are representative of 2 replicate experiments. D: Western blot detecting iNOS in total protein extracts from PC12 cells simultaneously treated for 24 hr with 10 ng/ml NGF and 10 ng/ml TNFα in medium containing serum, in serum free medium (SF) or in defined medium (N2). Results shown are representative of 3 replicate experiments. E: Western blot analysis detecting the presence of iNOS in total protein extracts from PC12 cells treated for 72 hr with 100 ng/ml IGF and 10 ng/ml TNF, individually or combined, as compared to cells simultaneously treated with 10 ng/ml NGF and 10 ng/ml TNFα or untreated controls (Cont). Results shown are representative of 4 replicate experiments.