Anti 4 hne

Mice were sacrificed three days after MPTP treatment and the SN was dissected out. SN lysates containing equal amounts of protein were loaded in each lane and separated in a 10-15% SDS-polyacrylamide gel, as described previously (Jin et al., 2011 (link)). Proteins were transferred to a nitrocellulose membrane and nonspecific binding sites were blocked with Licor Odyssey blocking buffer. The membranes were then incubated with different primary antibodies such as anti-IBA-1 (Abcam), anti-GFAP (EMD Millipore), anti-iNOS (Santa Cruz), anti-gp91phox (Abcam), anti-3NT (EMD Millipore) and anti-4HNE (R & D Systems). Next, membranes were incubated with one of the following secondary antibodies: Alexa Fluor 680 goat anti-mouse, Alexa Fluor 680 donkey anti-goat (Invitrogen) or IR dye 800 donkey anti-rabbit (Rockland). To confirm equal protein loading, blots were reprobed with a β-actin antibody (Sigma; 1:10000 dilution). Western blot images were captured with a Licor Odyssey machine (Licor, NE), and the bands were quantified using NIH ImageJ software.

Total cell lysates from primary rat neurons were subjected to standard Western blot assays, as previously described ^{20 (link), 31} . Protein concentrations from total neuronal lysates were determined by Bradford protein assay (Bio-Rad, Hercules, CA). Equal amounts of proteins were subjected to electrophoresis, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA). Membranes were probed with antibodies recognizing HO-1 (1:1000, Enzo Life Science, Farmingdale, NY) and β-actin (1:3000, Sigma-Aldrich, St. Louis, MO). After incubation in secondary antibodies (Santa Cruz Biotechnology, Dallas, TX), membranes were incubated with chemiluminescent substrates (Pierce, ThermoFisher Scientific, Pittsburgh, PA) and developed with X-ray film. ImageJ software was used for gel analyses.
For the Western slot blot assays, proteins were loaded directly onto PVDF membranes using a Bio-Dot Microfiltration apparatus (Bio-Rad, Hercules, CA). Anti-4-HNE (1:1000, R&D, Minneapolis, MN) and anti-tubulin (1:2000, Abcam, Cambridge, MA) were used as the primary antibodies ^{17 (link), 38 (link)}. The remaining procedures were the same as described above for the Western blots.