P jnk1 2

Magnolol, purity ≥98%, was purchased from Chengdu Reference Products (Chengdu, China). Dimethylsulfoxide (DMSO), oleic acid (OA), tyloxapol (Ty), and Oil Red O were obtained from Sigma-Aldrich (St. Louis, MO, USA). An Oil Red O stain kit (for cultured cells) was purchased from Solarbio Science & Technology (Beijing, China). Reactive oxygen species (ROS), TG, and total cholesterol (TC) assay kits were provided by Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). Human TNF-α enzyme-linked immunosorbent assay (ELISA) kits were provided by BioLegend (CA, USA). U0126, SB203580, SP600125, and LY290042 (specific inhibitors of ERK1/2, P38, JNK1/2, and AKT, respectively) and antibodies against AMPKα, P-AMPKα, AMPKβ, P-AMPKβ, adenosine ACC, P-ACC, P-ERK1/2, ERK1/2, P-JNK1/2, JNK1/2, P-P38, P38, IκB, P-IκB, and P-P65 were purchased from Cell Signaling Technology (Boston, MA, USA). AKT, P-AKT (Thr 308) and GAPDH antibodies were purchased from Affinity (OH, USA). Antibodies against P65, SREBP-1c and β-actin were purchased from Proteintech (Boston, MA, USA). PPARα and compound c (inhibitor of AMPK) were purchased from Abcam (Cambridge, MA, USA). HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies were provided by Boster (CA, USA). All other chemicals were of reagent grade.

The following antibodies were used for immunoblotting analyses: BACE1 (Millipore, AB5940, 1:500), pJNK1/2 (Cell Signaling Technology, #9251, 1:500); JNK1/2 (Cell Signaling Technology, #9252, 1:500); BAX (Santa Cruz Biotechnology, Sc-493, 1:100); Bcl-2 (Santa Cruz Biotechnology, Sc-509, 1:200); β actin (Sigma-Aldrich, A5441, 1:5000); Uch-L1 (Santa Cruz Biotechnology Sc-1183, 1:200).
Fresh frozen brains were homogenized in ice-cold buffer consisting of 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EGTA, 1 mM EDTA, 1% Triton™-X-100, 1 mM PMSF, phosphatase and protease inhibitors and then centrifuged at 12,000 rpm for 20 min at 4°C in order to obtain soluble proteins. Lysates (20 μg) were run on 4%–12% Tris-HCl gradient PAGE gel (Invitrogen) and then transferred to nitrocellulose blotting membrane (GE Healthcare 10600008). Peroxidase-conjugated secondary antibodies were incubated 1 h at RT and revealed with Luminata Forte Western substrate (WBLUF0100, Millipore). The correct protein loading was controlled normalizing with β actin antibody.

Western blot analyses were performed according to previous protocols [29 (link)]. Briefly, proteins were extracted from the cells or liver tissues by lysis buffer. Protein concentrations were determined, and the equal amounts of lysates were loaded, separated by SDS-PAGE gels, and transferred to PVDF membranes. The membranes were blocked and incubated with primary antibodies overnight at 4 °C followed by an incubation with HRP-conjugated secondary antibody. Signals were detected by an ECL system (GE Healthcare, Buckinghamshire, UK) and band intensities were quantified by densitometry using the Quality One software (Bio-Rad, Hercules, CA, USA). The primary antibodies against α-SMA (Cat#19245), CD36 (Cat#14347), P-JNK1/2 (Cat#4668), JNK1/2 (Cat#9252), P-NF-κB (Cat#3033), NF-κB (Cat#8242), and GAPDH (Cat#5174) were purchased from Cell Signaling Technology (Danvers, MA, USA), and the primary antibody against Col-1a1 (Cat#A1352) was purchased from ABclonal Technology (Wuhan, China).

Roswell Park Memorial Institute (RPMI) 1640 medium, penicillin-streptomycin (Pen Strep), fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Phorbol 12-myristate 13-acetate (PMA), LPS (Escherichia coli 055:B5), RIPA buffer, DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 1× Halt Protease and Phosphatase Inhibitor Cocktail was purchased from Pierce (Rockford, IL, USA). Human TNF-α and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, USA). Alamar blue reagent for cell viability assay was purchased from Life Technologies (Grand Island, NY, USA). Primary antibodies specific to COX-2, p-p38, p38, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, p-IκBᾳ, IκBᾳ, p-IKKᾳ/β, p-NFκBp65 and β-actin were purchased from Cell Signaling Technology (Beverly, MA) and, in addition, anti-rabbit secondary antibody conjugated to horseradish peroxidase was obtained from Cell Signaling Technology (Beverly, MA). Methanol and acetonitrile of HPLC grade were purchased from Fisher Scientific (Loughborough, UK). Phyllanthin, hypoPhyllanthin, niranthin gallic acid, ellagic acid, corilagin and geraniin (purity > 98%) were purchased from ChromaDex (CA, USA). Dexamethasone was obtained from CCM Duopharma Biotech Bhd (Selangor, Malaysia).

Total proteins in human aortic smooth muscle cell (HASMC) and aortic tissues were extracted by RIPA solution with phosphatase inhibitors and protease inhibitors, and western blot was performed as described previously (3 (link), 16 (link), 17 (link)). The antibodies used in this study included α-SMA (ab7817), LOX (ab174316), SM22 (ab14106), H3K4me1 (ab8895), H3K36me3 (ab9050), Bax (ab32503), and Bcl-2 (ab182858) which were bought from Abcam. LOXL1 (A10191) and LOXL4 (A13131) were got from ABclonal. LOXL2 (GTX105085), MMP2 (GTX634832), MMP9 (GTX100458), H3K9me3 (GTX121677), and PCNA (GTX100539) were purchased from GeneTex. LOXL3 (sc-377216) was obtained from Santa Cruz. β-actin (#8457S), Beclin-1 (#3495), S6 (#2317), phosphorylation of S6 (p-S6) (#5364), AKT (#4685), phosphorylation of AKT (p-AKT) (#4060), p38 (#8690), p-p38 (#4511), p-JNK1/2 (#9255), p-ERK1/2 (#4370), p-GSK3 β (#5558), H3K4me2 (#9725), H3K4me3 (#9727), p-p65 (#3033), cyclinD1 (#2978), and LC3 (#12741) were purchased from Cell Signaling Technology.