Anti iκbα

Lung tissues were homogenized by a homogenizer and lysed using RIPA buffer containing a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The protein concentration of each group was determined using BCA kits. Protein samples (30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis and subsequently transferred onto PVDF membranes, which were blocked by 5% skim milk for 1 hour at room temperature. After rinsing three times, the blots were incubated overnight at 4°C with the following primary antibodies: anti-TLR4 (1:1,000; Cell Signaling Technology), anti-NF-κB p65 (1:1,000; Cell Signaling Technology), anti-phospho-IκBα (1:1,000; Cell Signaling Technology), anti-IκBα (1:1,000; Santa Cruz), and anti-Cit-H3 (1:1000, Abcam). The membranes were washed three times, incubated with horseradish peroxidase–conjugated secondary antibodies (1:3,000; Sigma-Aldrich), and then visualized by an enhanced chemiluminescence kit (ECL plus). We used anti-GAPDH (1:2,000; Cell Signaling Technology), anti-β-actin (1:10,000; Sigma-Aldrich) and anti-Histone-H3 (1:1500; Sigma) as internal controls. To measure the relative ratio of protein expression, band intensities were quantified by Image-Lab software (Bio-Rad).

Protein samples at 10 μg were separated by 10 to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidenedifluoride membranes (Bio-Rad) by wet electroblotting. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature, incubated with anti-IκB-α (Santa Cruz Biotechnology), anti-p-AKT (Cell Signaling), anti-total AKT (Santa Cruz Biotechnology) and anti-Tubulin (Cell Signaling) antibodies overnight at 4 °C, and subsequently incubated with corresponsive horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies. Proteins were eventually visualized by utilization of an ECL kit (GE Healthcare) and normalized to the expression level of Tubulin.

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).

Tissue lysis, protein extraction, and Western blot analyses were performed as previously described27 (link). Proteins were dissolved in a lysis buffer and separated by SDS/PAGE for Western blot analyses. Primary antibodies included anti-phosphorylated NF-κB (p-p65), anti-NF-κB (p65), anti-β-actin (Cell Signaling Technology, Inc.), and anti-IκBα (Santa Cruz Biotechnology, Inc.). Secondary antibody was HRP-conjugated anti-rabbit IgGs (Pierce). The densitometric analyses of Western blotting images were performed using Image-Pro Plus software (Media Cybernetics).

Antibodies used were anti-RIPK1 (BD Biosciences, San Jose, CA, USA, #610459), anti-actin (MP Biomedicals, Solon, OH, USA, #69100), anti-IκB-α (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-371), anti-hRIPK3 (ThermoFisher Scientific Pierce, Waltham, MA, USA, PA1-41533), anti-mRIPK3 (Sigma-Aldrich, St Louis, MO, USA, #R4277 and IMGENEX, San Diego, CA, USA,, IMG-5523-2), anti-hMLKL (Genetex, Irvine, CA, USA, GTX107538), anti-mMLKL (Millipore, Billerica, MA, USA, #MABC604 and Abgent, San Diego, CA, USA, AP14272b-ev), anti-phospho-hMLKL (Abcam, Milton, Cambridge, UK, #187091), anti-hFADD (BD Biosciences, 610399), anti-mFADD (Millipore, 05-486), anti-thiophosphate ester (Epitomics, Burlingame, CA, USA, #2686-1), anti-Braf (ThermoFisher scientific, MA5-15495), anti-HPK1 (Cell Signaling, Danvers, MA, USA, #4472), anti-Hsp90 (Santa Cruz Biotechnology, sc-7947), anti-p38MAPK (Cell Signaling, #9212), anti-ERK1/2 (Cell Signaling, #9102), anti-Flag-HRP (Sigma-Aldrich, St Louis, MO, USA, #A8592) and anti-tubulin-HRP (Abcam, #ab21058). Secondary antibodies used were HRP-conjugated secondary antibodies against mouse, rabbit or rat immunoglobulin (GE Healthcare, Little Chalfont, Amersham, UK).

Cell culture reagents were obtained from Invitrogen (San Diego, CA, USA), unless otherwise specified. Fetal bovine serum (FBS) was from Biological Industries (Haemek, Israel) and oligofectamine and G418 from Invitrogen. NGF and BDNF from Peprothec (Rocky Hill, NJ). ProNGF from Scil Proteins GmbH (Halle, Germany) and ammonium pyrrolidinedithiocarbamate (PDTC) from Sigma-Aldrich (St. Louis, MO USA). Antibodies used in this study were the following: mouse anti-α-smooth muscle actin (α-SMA, 1∶500; Dako, Dakopatts, Denmark), anti-smooth muscle myosin (1∶400; NeoMarkers, Fremont, CA USA), anti-CD68 (Dako, 1∶250), anti-α-tubulin and anti-Chromosome Region Maintenance 1 (CRM1, 1∶1000; Sigma-Aldrich), rabbit anti-sortilin (1∶200, Abcam, Cambridge, UK; Chemicon Intern, Temecula, CA USA), anti-proNGF (Sigma-Aldrich), anti-bax protein (1∶200), anti-NF-κB p65 (1∶200), anti-p50 (1∶100), anti-IκB-α (1∶50), goat anti-p75^NTR (1∶200), anti-bcl-2 (1∶100, Santa Cruz Biotechnology, CA, USA) and anti-hypoxanthine-guanine phosphoribosyltransferase (HPRT; Abcam). Fluorocrome conjugated secondary antibodies were purchased from Jackson (Suffork, UK) and Invitrogen. Horseradish peroxidase-conjugated secondary antibodies were from Nordic (Tilburg, The Netherlands).