Antibodies against inos

Crocetin is donated by Tairui Biotechnology Co., Ltd (Nanyang, Henan, China). Crocetin was purified by HPLC with 98% purity, and it can be dissolved in DMSO (0.1% final concentration in culture medium). LPS (Escherichia coli Serotype 055:B5) was purchased from Sigma (St. Louis, MO, USA). AZD6244, SB202190, SP600125, and U0126 were from MedChemExpress (Monmouth Junction, NJ, USA). Antibodies against iNOS, phospho-ERK1/2, phospho-p38 kinase, phospho-JNK, ERK1/2, p38 kinase, JNK, and IκB-α were purchased from Cell Signaling Technology (Beverly, MA, USA). GAPDH and p65 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against HO-1 and Nrf2 were from Abcam (Cambridge, MA, USA); 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was from Beyotime Institute of Biotechnology (Beyotime, Guangzhou, China). Fetal bovine serum (FBS) was from Biological Industries (Kibbutz Beit Haemek, Israel). Murine macrophage-like RAW264.7 cells were purchased from ATCC (Rockville, MD, USA) and cultured at 37°C in a 5% CO₂ atmosphere in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS. Because FBS contains numerous compounds, such as LPS and growth factors, which influence the biological characteristics of macrophages [38 (link)–41 (link)], we performed all of the experiments under serum-free conditions.

Antibodies against Bcl-2 and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against iNOS, Bcl-xL, poly-ADP ribose polymerase (PARP), cleaved-PARP, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, E-cadherin, N-cadherin, vimentin, snail, and twist were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Nrf2 and Bax antibodies were purchased from Abcam, Inc. (Cambridge, MA, USA). Annexin V assay for apoptosis analysis was performed using the FITC Annexin V apoptosis detection kit I (BD Biosciences, San Diego, CA, USA). Transwell chambers with 8 µm pore size polycarbonate membrane inserts and Matrigel were obtained from Corning (Costar Corning Inc., NY, USA) and BD Biosciences (San Diego, CA, USA), respectively.

PACA was synthesized and characterized by NMR and MS as previously described [22 (link), 23 (link)]. Cell culture medium, fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against iNOS, cyclooxygenase-2 (COX-2) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH), and PPAR-γ was purchased from Cell Signaling Technology (Boston, MA, USA). The antibodies against Arg1, ED2 and CD163 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against ED1 and CD68 were purchased from Abcam (Cambridge, Massachusetts, UK). Anti-mouse CD80 FITC was obtained from BD Biosciences (San Diego, CA, USA). Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody were purchased from Life Technologies (Carlsbad, CA, USA). Anti-rabbit HRP-conjugated IgG secondary antibody and other chemicals including GW9662, a specific PPAR-γ inhibitor, were obtained from Sigma-Aldrich (St. Louis, MO, USA).