Anti phospho p65

LPS (Escherichia coli, O127:B8) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mas receptor antagonist A779 was obtained from AbBiotech (San Diego, CA, USA). ACE2 inhibitor MLN-4760 was a product from EMD Millipore (Darmstadt, Germany). SB203580 (a specific inhibitor of p38 MAPK), PD98059 (a specific inhibitor of ERK1/2) and SP600125 (a specific inhibitor of JNK) were purchased from Santa Cruz Biotechnology (Delaware, CA, USA). Rabbit anti-ACE2, anti-p50, anti-p65, and mouse anti-phospho-p50, anti-phospho-p65 and anti-IκBα antibodies were procured from Santa Cruz Biotechnology. Rabbit anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-/JNK, anti-phospho-JNK, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, and horse anti-mouse IgG antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). TNF-α and IL-1β kits were purchased from Invitrogen (Eugene, OR, USA). AngII and Ang-(1-7) enzyme-linked immunosorbent assay (ELISA) kits were from Kamiya Biomedical (Seattle, WA, USA).

After treatment for 2 h with or without etanercept (0.1 and 1 μg/mL) or adalimumab (1 and 10 μg/mL), cells were stimulated with the Th17-polarized panel and subsequently lysed with an equal volume of ice-cold lysis buffer (150 μl) 1 h later for MAPKs, p65-NFκB and STAT3 signalling and 24 h later for RORγt signalling. After centrifugation at 13,000 × g for 15 min, cell lysates were analysed by western blot with anti-MAPK (p38 and ERK), anti-phospho-MAPK (pp38 and pERK), anti-p65, anti-phospho-p65, anti-STAT3, anti-phospho-STAT3, and anti-RORγt antibodies (Santa Cruz Biotechnology, Santa Cruz, California, USA). The immunoreactive bands were visualized using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Sunnyvale, California, USA).

Cells and fresh frozen tumor tissues were homogenized in NP-40 lysis buffer (1% NP-40, 20 mM Tris–HCl pH 8, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 10 µg/mL aprotinin, 10 µg/mL leupeptin). Protein concentrations were determined using the Bradford protein assay (Bio-Rad Laboratories, Milano, Italy). Then, 30 µg protein/sample were separated by SDS-PAGE and blotted on a PVDF membrane. The following antibodies were used: anti-PTX3 (from B. Bottazzi, Humanitas Clinical Institute, Rozzano, Italy), anti-TLR4 (Bio-Rad), anti-phospho IRAK1 (Sigma-Aldrich, MO, USA), anti-phospho AKTser473 (Cell Signaling Technology, MA, USA), anti-phospho p65 (Santa Cruz Biotechnology, CA, USA). To normalize the amount of loaded proteins, all blots were probed with anti-β-actin (Sigma-Aldrich), anti-α-tubulin (Sigma-Aldrich), anti-GAPDH (Santa Cruz Biotechnology) or anti-HSC70 (Santa Cruz Biotechnology) antibodies. All primary antibodies were diluted 1:1000 and the secondary HRP-conjugated antibodies 1:5000. Chemiluminescent signal was automatically acquired by ChemiDoc™ Imaging System (Bio-Rad) at a final resolution of 62.2 pixel/mm².