Ab61392

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.

Interferon-gamma (IFN-γ, 300-02) and tumor necrosis factor alpha (TNF-α, 300-01A) were purchased from Peprotech. AMPK agonists Metformin hydrochloride (PHR1084), AICAR (A9978), Resveratrol (R5010); SIRT1 agonist SRT1720 (SRT1720) and NRF-2/HO-1 agonist Protoporphyrin IX cobalt chloride (C1900) were purchased from Sigma-Aldrich. Inhibitors of NF-κB Emodin (E7881) and JSH23 (J4455); IKKβ inhibitor IKK16 (SML1138); STAT1 inhibitor Fludarabine (F2773) and NOS2 inhibitor 1400W (W4262) were purchased from Sigma-Aldrich. Inhibitors of MEK1 and MEK2 PD98059 (ab120234), JNK SP600125 (ab120065) and MAPK SB202190 (ab120638) were purchased from Abcam. Reagents were dissolved in DMSO or water (Metformin and AICAR) at 100x stock concentrations and maximum DMSO concentration used in cells was 1%. Monoclonal antibody against 3-nitrotyrosine (ab61392) was purchased from Abcam. Monoclonal antibody against NF-κB p65 (sc-8008) was purchased from Santa Cruz Biotechnology.

Cardiac left ventricles were homogenised using an SDS-based lysis buffer containing protease inhibitor cocktail (1 tablet per 10 mL lysis buffer, Roche, Herts, UK), and protein concentration determined using the colorimetric Lowry copper based protein assay protocol [31 (link)]. Samples were loaded onto a 12% Tris-SDS-polyacrylamide gel for protein separation according to size, after which proteins were transferred onto a PVDF membrane. The membrane was treated with 5% w/v BSA for 1 h at room temperature to block unbound pores. Membranes were incubated with primary antibody, either at room temperature for 1 h or overnight at 4 °C, and species-specific horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Chemiluminescence was detected by incubating membranes with 1 mL enhanced chemiluminescence substrate (Merck Millipore, London, UK) for 1 min before developing membranes in the G-Box gel documentation system (G-Box, Syngene, Cambridge, UK) and images captured using GeneSnap software (version 7.12, Syngene, UK). Primary antibodies against gp91(phox) (611414, BD Biosciences, Berks, UK), HO-1 (ab13243, Abcam, Cambridge, UK), nitrotyrosine (ab61392, Abcam, UK), and GAPDH (AM4300, Ambion, Oxford, UK) were used in this study. Densitometry analysis was performed using ImageJ software (version 1.48), and normalised to the loading control GAPDH.

Endogenous peroxidases were quenched (3% hydrogen peroxide in methanol) followed by blocking (4% normal horse serum) for one hour in a humidified chamber. Sections were then incubated with mouse monoclonal anti-nitrotyrosine antibody (1:500, ab61392, Abcam) overnight at 4 °C. The Ultra-Sensitive ABC Peroxidase Mouse IgG Staining Kit (Thermo Fisher Scientific) was used for the application of the secondary antibody as well as the ABC complex. Sections were developed with 3–3′-diaminobenzidine (Sigma-Aldrich) and counterstained with hematoxylin for imaging using light microscopy. Images were captured using a Nikon Eclipse e400 microscope outfitted with a Nikon Coolpix 990 digital camera. Using the 40 × /0.65 N.A. air objective lens, sequential images of the tissue sections were taken to capture the entire length and depth of the articular cartilage across both the medial and lateral femoral condyles/tibial plateaus.