Horseradish peroxidase conjugated anti rabbit or anti mouse igg secondary antibody

Total protein was obtained using radioimmunoprecipitation assay (RIPA) buffer (Pierce) on ice. After centrifugation at 13 000 g for 15 min at 4 °C, the lysate was heated at 95 °C for 5 min in SDS loading buffer. The samples were then separated on 6% or 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes by a wet transfer apparatus (Bio-Rad). The membranes were blocked with 5% skim milk powder at room temperature for 1 h and incubated with primary antibodies overnight at 4 °C. The primary antibodies used include: rabbit anti-USP34 (1:2 000; A300-824A; Bethyl Laboratories), rabbit NFIC antibody (1:3 000; 16399-1-AP; Proteintech), mouse OSX antibody (1:100; sc-393325; Santa Cruz Biotechnology), mouse DSPP antibody (1:100; sc-73632; Santa Cruz Biotechnology), rabbit α-tubulin antibody (1:5 000; 11224-1-AP; Proteintech), mouse FLAG antibody (A8592; Sigma), rabbit anti-HA (1:1 000; 3724; Cell Signaling Technology). The PVDF membranes were then incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Cell Signaling Technology) and visualized using Immobilon reagents (Millipore).

Cells were washed with 1X PBS and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (GeneTex Inc., Hsinchu City, Taiwan). Twenty-five μg of extracted protein was separated by SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Merckmillipore, Danvers, MA, USA). The membrane was then incubated with primary antibodies at 4 °C overnight. After washing with Tris-buffered saline (TBS)/0.05% Tween-20, the membrane was then incubated with specific secondary antibody conjugated with horseradish peroxidase (HRP) at room temperature for one hour. The signals were then developed by incubation with chemiluminescence substrate (PerkinElmer Inc.) and captured with the Luminescence-Image Analyzer (FUSION SOLO, Vilber Lourmat Deutschland GmbH, Germany). The antibodies used in this study were listed as followed: rabbit IgG anti-TRIB3 was purchased from Proteintech Group, Inc. (Rosemont, IL, USA); rabbit IgG anti-Notch1 was purchased from Abcam (Cambridge, UK); rabbit IgG anti-c-Myc antibody was purchased from GeneTex Inc. Mouse IgG anti-BCLAF1, mouse IgG anti-BNIP1, and mouse IgG anti-DDX5 antibodies were purchased from Santa Cruz Biotechnologies, Inc. (Dallas, TX, USA). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies were purchased from Cell Signaling Technology, Inc. (Boston, MA, USA).

Linarin and rosmarinic acid were purchased from Must Bio-technology Co. (Chengdu, China). Lipopolysaccharides and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Dulbecco’s modified eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Corning Inc. (Corning, NY, USA). Nitric oxide assay kit was purchased from Beyotime Biotech. (Jiangsu, China). Mouse TNF-α, IL-1β, and IL-6 ELISA kits were purchased from eBioscience, Inc. (San Diego, CA, USA). Trizol, RevertAid™ first strand cDNA synthesis kit, power sybr green pcr master mix, and Super Signal^TM West Pico Chemiluminescent Substrate were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Oligonucleotide primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Horse-radish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies were obtained from Cell Signaling Technology (Danvers, CO, USA). Fluorescein-conjugated affinipure goat anti-rabbit IgG was purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). Antibodies against phospho (p)-ERK, p-p38, p-JNK, p-Akt, Akt, p-p65, and p-IκBα were obtained from Cell Signaling Technology. All other reagents were of analytical grade.