Anti phospho jnk

Letrozole (4,4-(1H-1,2,4-Triazol-1-ylmethylene) bisbenzonitrile) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). Recombinant RANKL and M-CSF were obtained from PeproTech (Rocky Hill, NJ, USA), and the antibodies against NFATc1 and c-Fos were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The other antibodies (anti-phosphor-IKK, anti-IKK, anti-phosphor-ERK, anti-ERK, anti-phospho-JNK, anti-JNK, anti-phosphor-p38, and anti-p38) were purchased from Cell Signaling Technology (Danvers, MA, USA). The leukocyte acid phosphatase kit (TRAP staining kit) and antibody for β-actin were obtained from Sigma-Aldrich. All other reagents were purchased from Sigma-Aldrich.

The LV tissue was lysed in radioimmunoprecipitation assay (RIPA) lysis buffer, and the total protein was extracted and detected with a BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). Approximately 30 μg of total protein was separated by electrophoresis on Laemmli sodium dodecyl sulfate (SDS) polyacrylamide gels. After electrophoresis, the samples were transferred to Immobilon-FL PVDF membranes (Millipore, USA). The membranes were blocked with 5% nonfat milk and then incubated with anti-IL-22, anti-IL-22R1 (both from Abcam, Cambridge, England), anti-atrial natriuretic peptide (ANP), anti-B-type natriuretic peptide (BNP), anti-β-myosin heavy chain (β-MHC, triple from Santa Cruz, Dallas, TX, USA), anti-STAT3, anti-phospho-STAT3, anti-ERK, anti-phospho-ERK, anti-c-Jun N-terminal kinase (JNK), anti-phospho-JNK, anti-P38, anti-phospho-P38, and anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH, nine above from Cell Signaling Technology, Boston, USA) antibodies at 4°C overnight. The secondary antibodies were incubated at room temperature for 1 hour. The blots were scanned using a two-color infrared imaging system (Odyssey; LI-COR Biosciences, Lincoln, NE, USA).

Dulbecco’s modified Eagle medium and penicillin–streptomycin solution were purchased from GIBCO (Grand Island, NY, USA). Fetal bovine serum was provided by Hyclone (Logan, UT, USA). Monoclonal rabbit antibodies, including anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK, anti-phospho-JNK, anti-p65, anti-phospho-p65, NLRP3, and cleaved caspase-1 were obtained from Cell Signaling Technology (Boston, MA, USA). Receptor for AGE (RAGE) monoclonal rabbit antibody was purchased from AbCam (Cambridge, MA, USA). HRP-marked anti-β-actin antibody was supplied by Biorad (San Diego, CA, USA). 4′-Methoxyresveratrol (3,5-dyhydroxy-4′-methoxylstilbene) was purchased from Great Forest Biomedical (Hangzhou, China). BSA was obtained from ABCONE (Shanghai, China). KI and acetic acid were obtained from Sangon Biotech (Shanghai, China). Methylglyoxal, 2’,7’-dichlorodihydrofluorescein diacetate and other reagents were purchased from Sigma (St. Louis, MO, USA).

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).

Total protein extracts or cytoplasmic and nuclear fractions were analyzed by SDS-PAGE and transferred onto a PVDF membrane (Roche Applied Science, Switzerland). Blots were blocked with PBS containing 5% non-fat dry milk and 0.1% Tween and then incubated with primary antibody. Primary antibodies were mouse anti-flag and anti-V5 antibodies (Invitrogen, USA); mouse anti-actin, rabbit anti-hepsin, and normal IgG antibodies (Santa Cruz Inc, USA); anti-phospho-p38, anti-phospho-C/EBP-β, anti-phospho-ERK, anti-phospho-JNK, anti-p38 anti-C/EBP-β, anti-ERK, anti-JNK, anti-p65, anti-p50, anti-histone H3 and anti-α-tublin (Cell Signal Technology, USA); anti-C3, anti-HBc and anti-HBx antibody (Abcam, UK).

Carnosic acid, 5-aminosalicylic acid (5-ASA), sodium carboxymethyl cellulose, haematoxylin and eosin were purchased from Sigma Aldrich (St. Louis, MO). Dextran sodium sulfate was purchased from MP Biomedicals (Solon, OH). Anti-p65 (#8242), anti-phospho-p65 (#3039), anti-IĸBα (#9242), anti-phospho-IĸBα(#2859), anti-Stat3 (#4904), anti-phospho-Stat3 (#9145), anti-JNK (#9252), anti-phospho-JNK (#4668), anti-c-Jun (#2315), anti-phospho-c-Jun (#2361), anti-NLRP3 (#15101), anti-ASC (#67824), anti-Ubiquitin (#3933), iNOS (#13120), anti-H3k27Me3 (#9733), anti-H3k4Me3 (#9751) and anti-β-actin (#4970) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-Keap1 (ab150654), anti-Nrf2 (ab62352) and Cullin3 (ab75851) antibodies were purchased from ABCAM. Anti-caspase1 (sc-56036) antibody was purchased from Santa Cruz Biotechnology, TRIzol reagent, TaqMan primers and TaqMan PCR Master Mix were purchased from Invitrogen (Carlsbad, CA).