Fig. 3. Overexpression of iNOS results in nitration of α-synuclein and aggregation in vitro.

(A) Western blot showing differentiated PC12 neuron lysates (15 μg/well) after infection with AAVs before immunoprecipitation (IP) probed with antibodies against α-syn, iNOS, or α-tubulin (top panel) and after IP with an anti-α-syn antibody (syn42) probed with a nitrated α-syn specific (Nsyn12) antibody (bottom boxed panel). The IP membrane was then reprobed with syn42 to verify IP. Graph shows densitometric analysis (Image J) as ratio of N-α-syn/total α-syn monomers (Nsyn12/syn42). (B) AFM measurements from supernatants from 293 cell cultures with recombinant α-syn added to the supernatants for 48 hrs (Left graph) +/- iNOS expression. N-α-syn control was aggregated in vitro prior to addition to media. Data represent the mean ± SEM of 52 measurements/treatment group. *p < 0.001 compared to cells in the absence or presence of α-syn. The right graph is the AFM measurements from recombinant α-syn starting materials (before addition to cell cultures) either unmodified or nitrated and aggregated. *p < 0.001 compared to height of unmodified α-syn. Representative images of α-syn/particles in supernatants are shown. (C) Western blots showing supernatants (25 μl/ lane) from the AFM experiment detected with syn42 α-syn antibody (Left blot). Increased presence of oligomers indicated by arrowhead. Also shown is detection of starting materials (recombinant proteins, 15 μg/well) with syn42 antibody. (D) NO concentration (nitrite) in the 293 supernatants taken for AFM. Data represent the mean ± SEM of 4 measurements/treatment group. *p < 0.001 compared to cells only.