Ecl substrate

VICs were washed two times in PBS, solubilised and homogenised in RIPA buffer solution (Sigma) supplemented with protease inhibitor cocktail 1X (Roche). Cells were scraped with a rubber policeman; the lysate was transferred into a 1.5ml microtube and vortexed. Proteins were quantified with a Pierce BCA protein assay (Thermo Scientific) after a 10,000g centrifugation for 10 minutes at 4°C. Total protein homogenates (7.5 μg) were denatured and separated on 10% Bis-Tris gels (Invitrogen). Electrophoretically resolved bands were then transferred on nitrocellulose membranes (Hybond C, Amersham). Membranes were blocked for 1 hour in Phosphate-Buffered Saline (PBS) containing 0.1% Tween-20 (PBS-T) and 5% (w/v) non-fat powdered milk. Then, they were incubated for 1 hour with primary antibodies (Table 1) in PBS-T containing 5% (w/v) non-fat powdered milk. Membranes were then washed three times in PBS-T and incubated with corresponding horseradish peroxidase conjugate secondary antibody (Table 1) for 1 h at room temperature in PBS-T. Membranes were washed five times in PBS-T. Visualization of the protein bands was accomplished using enhanced chemiluminescence (ECL) substrate (Amersham) and positivity was captured on Hyperfilm (Amersham). Films were scanned and bands were quantitated using the QuantityOne program (Biorad). Levels of expression were normalised to GAPDH.

The cellular proteins from cell suspension were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12% acrylamide). After electrophoresis, proteins were transferred onto PVDF membranes (Millipore). The PVDF membrane was soaked in 5% milk before incubation with swine anti-PCV2 serum (1:100) overnight at 4 °C. Detection was achieved with a peroxidase-conjugated goat anti-swine Ab (KPL), followed by the addition of an ECL substrate (Amersham) and Chemiluminescence imaging was taken by ChemiDoc Touch (Bio-Rad, USA).

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).