Horseradish peroxidase conjugated anti mouse secondary antibody

GST-MinD (5 μM) was incubated with His-cyto-AtoS (5 μM) in buffer-B (50 mM Hepes pH 7.4, 150 mM KCl) at room temperature for 30 min. HisPure cobalt resin was added to the reaction mixture and incubated for 1 h at 4 °C. His-cyto-AtoS (5 μM) or GST-MinD (5 μM) alone were used as controls. The whole reaction mixture was transferred to a spin column, washed three times with buffer B containing 20 mM imidazole and eluted with buffer containing 50 mM Hepes pH 7.4, 150 mM KCl, and 300 mM imidazole. The GST-tagged MinD fraction eluted along with His-cyto-AtoS was verified on a 12% SDS-PAGE and also subjected to Western blot analysis. Anti-GST (Invitrogen: 136700), anti-His primary antibodies (Sigma), and anti-mouse-horseradish peroxidase–conjugated secondary antibodies (Sigma) were used for the detection of MinD and cyto-AtoS. All antibodies were used at a 1:10,000 dilution.

Mice ear skin tissues were collected 30 min post‐treatment of vehicle and cinnamaldehyde, and snap frozen at −80°C until processing. Tissue was then lysed in SDS lysis buffer containing protease (1 tablet per 50 mL, Roche) and phosphatase (1 tablet per mL, Roche) inhibitor. Lysates were clarified by centrifuging at 2600 g for 10 min at 4°C. Protein concentration was assessed using the Bradford dye‐binding method kit (Bio‐Rad). Fifty micrograms of protein was loaded and separated by SDS‐PAGE and transferred to PVDF membranes using a semi‐dry technique (Aubdool et al., 2014). Membranes were blocked with 5% milk in PBS containing 0.1% Tween and incubated with primary antibodies against nitrotyrosine (1:500 dilution, Abcam ab61392) (Smillie et al., 2014) and loading control β‐actin (1:2000, Sigma A1978) dilution in 3% BSA in PBS and 0.1% Tween for 16 h at 4°C. Membranes were washed further with PBS 0.1% Tween and incubated with a horseradish peroxidase conjugated anti‐mouse secondary antibody (1:2000/5000 dilution, Sigma). Proteins were detected by enhanced chemiluminescence (Piercenet, UK) and densitometric analysis performed using Image J analysis software (NIH, USA). Total nitrotyrosine signal was calculated and normalized to the loading control β‐actin.

Male Sprague-Dewley rats with body weights of 240-350 g were provided by the Medical Experiment Animal Center of Ningxia Medical University. All animal usage and procedures were in strict accordance with the Chinese Laboratory Animal Use Regulations. Efforts were made to minimize animal stress and to reduce the number of rats used for this study.
Polyclonal anti-GFAP antibody (Santa Cruz), monoclonal anti-NeuN antibody (Sigma), polyclonal anti-ICAM-1 antibody (Protect), polyclonal anti-β-actin antibody (Sigma), horseradish peroxidase-conjugated anti-mouse secondary antibody (Sigma), and streptozotocin (STZ, Calbiochem, Germany) and the ICAM-1 In Situ Hybridization Detection kit were purchased from Boster Biotechnology Co (Wuhan, China).

[7,8-³H]Dopamine (21.2 Ci/mmol (0.784 TBq/mmol)) and [1-7,8-³H]noradrenaline (12.1 Ci/mmol (0.448 TBq/mmol)) were purchased from Perkin Elmer, Monza, Italy. Nicotine hydrogen tartrate salt, choline, 4-aminopyridine, 6-nitroquipazine, desipramine, beta-amyloid (40-1), horseradish peroxidase-conjugated anti-mouse secondary antibody, Ponceau Red were from Sigma (Sigma-Aldrich, St Louis, MO, USA); Monoclonal antibody 6E10 was from Covance ImmunoTechnologies, Dedham, MA; 5-iodo-A-85380, dihydro-β-erythroidine, α-conotoxin MII, oxotremorine sesquifumarate, desformylflustrabromine hydrochloride, galantamine hydrobromide, SR 165845, beta-amyloid (1-40) were from Tocris (Tocris Bioscience, Bristol, UK).