Mpges 1

Hind paws were homogenized in liquid N₂ with a phosphate buffer according to Rosillo et al., (2014), in order to isolate cytoplasmic and nuclear proteins [31 (link)]. Protein concentration was determined with Bradford’s colorimetric method [33 (link)]. Nuclear fraction was used to measure p50 and p65 expression and, cytoplasmic fraction was used for the rest of determinations. A total of 50 μg of proteins were separated on 10% sodium dodecyl sulphatepolyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and, incubated overnight at 4 °C with specific primary antibodies: COX-2, iNOS, pJNK, JNK, pp38, p38, pERK 1/2, ERK 1/2, Nrf-2, pSTAT-3, IκB-α, p50, p65 (Cell Signaling Technology^®, Danvers, MA, USA), HO-1 (Henzo^®, Madrid, Spain), mPGES-1 (Abcam^®, Cambridge, MA, USA). Membranes were incubated for 2 h at room temperature with horseradish peroxidase-labeled secondary antibody antirabbit (Cell Signaling Technology^®, Danvers, MA, USA) or antimouse (Dako^®, Atlanta, GA, USA). Blots were analyzed with β-actin antibody (Abcam^®, Cambridge, MA, USA) to prove equal loading. The immunosignals were captured with Amersham Image 600 from GE Healthcare (Buckinghamshire, UK) and signals were analyzed and quantified using Image Processing and Analysis in Java (ImageJ, Softonic).

Cells at different confluencies were washed in PBS and fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min. For permeabilization, the cells were incubated with 0.05% Triton X-100 solution at room temperature for 10 min and blocked with 5% normal goat serum (NGS) at room temperature for 1 hour. Then, the cells were stained with specific primary antibodies against COX-2 and mPGES-1 (Abcam, Cambridge, MA, USA) followed by 2 hours of incubation with Alexa 488-labelled secondary antibody (1:1,000; Molecular Probes, Eugene, OR, USA). The nuclei were stained with DAPI. The images were captured by a confocal microscope.

Magnolin was supplied by Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against IL-1β was obtained from the Invitrogen Life Technologies (Carlsbad, CA, USA). Anti-MMP-3, -8, -9, -13, mPGES-1, and TNF-α antibodies were purchased from Abcam Inc. (Cambridge, CA, USA). Anti-MMP-1, pTAK1, phospho-ERK, ERK, phospho-JNK, JNK, NF-κB pathway antibody sampler kit and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat anti-Mouse lgG-HRP and Goat anti-Rabbit lgG-HRP were purchased from GenDEPOT (Barker, TX, USA).