Ez western lumi pico

The total protein of ASCs or macrophages was extracted using sample buffer [62.5 mM Tris-HCl, pH 6.8, 34.7 mM sodium dodecyl sulfate (SDS), 5% β-mercaptoethanol, and 10% glycerol], and equal volumes of the protein samples were separated by 10% or 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membrane was blocked with 5% skim milk or 5% bovine serum albumin in Tris-HCl-buffered saline containing 0.05% Tween 20 (TBST) for 30 min, and incubated with primary antibodies against TSG-6, Cox-2, IDO2, and GAPDH (Santa Cruz Biotechnology, 1:1000); pp38, SMAD2/3, and cIL-1β (Cell Signaling Technology, 1:1000); and pSmad2/3 (Thermo Fisher Scientific) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibodies (Cell Signaling Technology, 1:5000) for 1 h at room temperature. After primary and secondary antibody incubation, the membranes were washed thrice for 5 min with TBST and then treated with an EZ-Western Lumi Pico or EZ-Western Lumi Femto (DoGenBio, Seoul, Republic of Korea). The target bands were detected using a ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA).

Protein samples were obtained from cells lysed with chilled ProEXTM CETi protein extract solution (Translab, Daejeon, Korea). Denatured samples were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Then, the proteins were transferred onto nitrocellulose (NC) membranes (Bio-Rad, Hercules, CA, USA) [30 (link)]. The membranes were incubated overnight at 4 °C with primary antibodies such as anti-PARP, anti-cleaved caspase-3, anti-Bip, anti-CHOP, anti-p-JNK, anti-E-cadherin, anti-N-cadherin (Cell Signaling Technology, Beverly, MA, USA), anti-p53, anti-p21, anti-Bax, anti-Bcl2, anti-GAPDH, anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-IRE1α (Abcam, Cambridge, MA, USA). After the membranes were incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibodies, the proteins were detected using EZ-Western Lumi Pico (DoGenBio).

The cells were seeded in 60 mm dishes for 24 h and then pre-treated with different concentrations of TVE for 1 h; all dishes, except the control, were subsequently stimulated with H₂O₂ for 10 min. Cells were washed twice with phosphate-buffered saline, harvested, washed, and lysed in radioimmunoprecipitation assay buffer. The protein content of the cell lysates was measured using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal volumes of lysates were electrophoresed on 4–12% Bis-Tris protein gels and transferred to nitrocellulose membranes. Primary antibodies used were as follows: anti-phosphorylated (phospho)-p38, anti-p38, anti-phospho-extracellular signal-regulated kinase (ERK), anti-ERK, anti-phospho-c-Jun N-terminal kinase (JNK), anti-JNK, anti-phospho-Akt, anti-Akt, and anti-β-actin. The secondary antibodies used were anti-rabbit and anti-mouse IgG. Antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA), Santa Cruz Biotechnology (Dallas, TX, USA), and Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were detected using a chemiluminescence detection kit (EZ-Western Lumi Pico; DoGen Bio, Seoul, Republic of Korea) on a Davinch–Chemi Imaging System (CAS400SM; Davinch-K, Seoul, Republic of Korea). Expression levels were quantified using ImageJ version 1.53t (National Institutes of Health, Bethesda, MD, USA).