Hrp conjugated secondary antibody

We used NuPAGE Novex 4%–12% Bis-Tris gradient gels (Life Technologies), and ran them using the manufacturer’s protocol with a reducing MES SDS buffer system. Protein transfer to nitrocellulose for blotting was performed using the iBlot system (Life Technologies), with a standard (P0, 8 mins) protocol. Nitrocellulose was washed briefly, and then blocked for 30 mins in 5% w/w non-fat dried milk (Marvel) in Tris buffered saline/0.05% Tween-20 (TBST). Membranes were then incubated in antibody diluted in blocking buffer (5% milk, TBST) overnight at 4°C. The following day, membranes were washed for 10 min three times (in TBST) and then incubated with 1:10,000 HRP-conjugated secondary antibody (Sigma-Aldrich) diluted in blocking buffer for 60 min. Three more 10 min TBST washes were then performed before chemiluminescence detection using Immobilon reagent (Millipore) and imaging using a Gel-Doc XR⁺ system (Bio-Rad). Quantification was performed using Image Lab Software 6.0 (Bio-Rad).

Equal amounts of proteins from FhNEJ-Som samples used for mass spectrometry analysis were separated by SDS-PAGE in 12% gels and transferred to a nitrocellulose membrane. Total protein staining was performed with Sypro Ruby (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions followed by incubation for one hour at room temperature in a blocking solution of PBST containing 2% BSA. F. hepatica cathepsin B1-3 anti-serum produced in rabbit was diluted 1:500 in blocking buffer and added to the blot overnight at 4 °C. After incubation with HRP-conjugated secondary antibody (Sigma, St. Louis, MO, USA) diluted 1:2000 in blocking buffer, protein bands were detected using enhanced chemiluminescence (Clarity Western ECL Substrate, BioRad, Hercules, CA, USA) on a ChemiDoc MP Imaging System (BioRad, Hercules, CA, USA).

Transfected DF-1 cells were collected by centrifugation, an appropriate amount of RIPA buffer lysate was added for lysis for 5 min, and the protein concentrations were determined using a BCA protein assay kit (Beyotime Biotechnology, Beijing, China). The proteins were denatured by heating, separated on 10% SDS-PAGE gels, and transferred to a nitrocellulose membrane (Millipore, Boston, MA, USA). The membranes were blocked with 5% skimmed milk in PBS containing 0.1% Tween 20 (PBST) for 1 h at room temperature and incubated with mouse anti-VP1 monoclonal antibody or rabbit anti-VP2 polyclonal antibody at 4 ℃ overnight. The blots were washed 3 times with PBST and incubated with HRP-conjugated secondary antibody (Sigma, St. Louis, MO, USA) for 1 h at room temperature. The blots were washed, and positive reactions were detected with an enhanced chemiluminescence (ECL) detection system (Beyotime Biotechnology, Beijing, China).

The expression level of Nrf2 was determined by western blot analysis. Cells were collected and cytosolic and nuclear proteins were extracted using a commercial nuclear and cytoplasmic protein extraction kit (Beyotime Biotechnology, Beijing, China) according to the manufacturer's instruction. The proteins were denatured by heating in the water bath at 95°C for 5 min, and separated on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore, USA). The membranes were blocked with 5% skim milk dissolved in PBST at room temperature for 1 h and incubated with anti-Nrf2 and anti-Lamin B1 mAbs (Abcam, Cambridge, MA) at 4°C overnight. The membranes were washed with PBST for three times and incubated with an HRP-conjugated secondary antibody (Sigma, USA) for another 1 h at room temperature. After washing with PBST three times again, positive reactions were detected by an enhanced chemiluminescence (ECL) detection system (Beyotime Biotechnology, Beijing, China). The band intensities were quantified using a VILBER Fusion FX5 Luminescence image analysis system (Vilber Lourmat, France) and compared with different groups.

Proteins were separated by SDS/PAGE and transferred to a PVDF membrane. Membranes were blocked for 1 h in TBST + 5% milk, followed by overnight incubation at 4 °C with primary antibody in blocking solution. The next day, membranes were washed 3× with TBST, followed by incubation at room temperature for 1–2 h with HRP-conjugated secondary antibody (Sigma) in blocking buffer. After three washes with TBST, Western blots were imaged using ECL Plus reagent (Pierce) on a GE ImageQuant LAS4000. Antibodies used in this study include: mouse anti-GRA1 (1:1,000 dilution; BioVision), mouse anti-GRA2 (1:1,000 dilution; BioVision), mouse anti-GRA3 (gift of J.-F. Dubremetz, University of Montpellier, Montpellier, France; 1:2,000 dilution), mouse anti-GRA4 (gift of L. D. Sibley; 1:2,000 dilution), mouse anti-GRA5 (1:1,000 dilution; BioVision), mouse anti-GRA6 (gift of L. D. Sibley; 1:4,000 dilution), rabbit anti-GRA7 (gift of L. D. Sibley; 1:5,000 dilution), rabbit anti-ROP2 (1:10,000 dilution), rat anti-HA (1:1,00 dilution; Sigma). Western blots for quantification were performed independently with each antibody to avoid residual signal due to incomplete stripping.