P smad2 3

Ten μg each of ES-P and F3 fraction (Fig 3) or 10 μg ES-P and total extract from T. spiralis, TS 15-1c (Fig 5B) or 10 μg of purified TS 15-1c antibody (Fig 5C) or 15 μg of each ear tissue samples (Fig 7) were separated on 10% acrylamide SDS-PAGE gel at 100 V for 90 min. Sweden), The loaded proteins were transferred onto a nitrocellulose membrane (Amersham Biosciences, Little Chalfont, UK) and blocked with 5% skim milk in TBST at 4°C overnight. Then, the membrane was incubated with primary antibody (polyclonal α-F3, α-TS 15-1c (1:500); time-course sera (1:1,000), α-TGF-β1 (1:1000; abcam, Carlsbad, CA, USA),;p-Smad2/3 (1:1000; Thermofisher science, Waltham, MC, USA),;α-mouse type I collagen (1:1000; abcam), and actin (1:5000, abcam)) in 5% skim milk in TBST for 2 hrs at room temperature. The secondary antibody, α-mouse or α-rat IgG-HRP conjugate (Sigma, Seoul, Korea) was used at 1:5,000 dilution for 1 hr at room temperature. HRP was detected using an ECL substrate (Amersham Biosciences, Uppsala, Sweden), analyzed using the LAS 3000 machine. (Areas of the detected bands were determined and compared by Image J software).

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).