Versadoc system

Liver (100 mg) (n = 5 − 6) was homogenized and assayed as before [11 (link), 12 (link)]. The secondary antibodies were anti-mouse (GE Healthcare, UK), anti-rabbit, and anti-goat (Bio-Rad, USA). Membranes were revealed using ECL substrate in a VersaDoc system (Bio-Rad, USA) and analysed with ImageQuant® (Molecular Dynamics, USA).

Aliquots of 100 μl of individual E. granulosus HF samples were resolved on SDS-PAGE 12% and electrophoretically transferred onto a nitrocellulose membrane. A pool of rabbit polyclonal antibodies raised against each recombinant AgB subunit (AgB8/1 to 5) were used at 1:70.000 dilution as primary antibody. The pool of antibodies corresponds to the IgG fraction of each subunit specific serum purified using HiTrap ProteinG HP columns (GE Healthcare), according to the manufacturer's protocol. A horseradish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare) diluted at 1:7.000 was used as the secondary antibody. Blots were developed using the chemiluminescent reagent ECL Plus (Pierce, ThermoScientific) and imaged in VersaDoc system (BioRad). HF samples with higher AgB content were used for the protein purification step (S1A Fig).

For Western blot analysis, 1 × 10⁶ Cos-7 cells were seeded in 6-well plates during 24 h and then treated with EA-Aa (500–1,000 μg·mL⁻¹) for 24 h. After, cells were washed with ice-cold PBS, disrupted in lysis buffer (0.25 M Tris-HCl, 125 mM NaCl, 1% Triton-X-100, 0.5% SDS, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 2 mM Na₃VO₄, 10 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 10 mM PMSF, and 40 μL of protease inhibitor), subjected to three freeze/thaw cycles in liquid nitrogen, and centrifuged (14,000 rpm, 20 min, 4°C). Protein concentration of the supernatant was measured through the BCA Protein Assay Kit [41 (link)], and the samples were mixed with Laemmli buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.01% bromophenol blue). An amount of 15 μg of total protein was loaded onto 8% SDS-PAGE, solved and electroblotted onto PVDF membrane. A rainbow marker was used as a standard weight protein marker. Membranes were blocked with TBS-T 0.01% and 5% BSA and incubated with primary antibody (against Sirt1, ERK, phospho-ERK [Thr202/Tyr204], Nrf2, phospho-Nrf2 [Ser40], and catalase) overnight and with secondary antibodies (anti-rabbit, anti-mouse, and anti-goat) for 2 h at room temperature. Calnexin was used as the loading control. Immunoblots were detected with an ECL substrate and the Versadoc system (Bio-Rad Laboratories, USA).

Protein expression was determined by performing Western blot analysis [32 (link)]. Whole-cell lysates or membrane fractions were separated by 10% Tris-Glycine SDS-PAGE. Following proteins electrophoretic transfer into nitrocellulose membrane, blots were blocked with 5% skim milk in TBS-T buffer (5% Tween 20) for 1 h. Primary monoclonal antibodies against TNFα, β-actin, Na⁺K⁺ATPase (Sigma-Aldrich, St. Louis, MO, USA), and CB1 and CB2 (Santa Cruse Biotechnology, Santa Cruz, CA, USA) were used at a concentration of 1:1000. The host of antibodies against TNFα and β-actin was mouse, while in the case of CB1, CB2 and Na⁺K⁺ATPase, the host was rabbit. Secondary antibodies labeled with alkaline phosphatase were used at a concentration of 1:1000 (Sigma-Aldrich, St. Louis, MO, USA). Protein bands visualization was made using the BCIP/NBT Liquid substrate system. The Versa Doc System and Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA, USA) was applied for bands quantification. Level of each protein was expressed as the percentage of the protein intensity obtained in the control cells.

Tissues were homogenized as previously described and samples were loaded in 8% polyacrylamide gels, separated by SDS-page, and transferred to a PVDF membrane (Advansta, San Jose, CA, USA). Membranes were incubated with the specific primary antibodies overnight at 4 °C (listed below), and then incubated for 2 h at room temperature with secondary antibodies. The secondary antibodies were anti-mouse (GE Healthcare, Chicago, IL, USA, EUA) and anti-rabbit (Bio-Rad, Des Plaines, IL, USA). Membranes were revealed using ECL substrate in a Versadoc system (Bio-Rad, Des Plaines, IL, USA) and analyzed with Image Quant^® (Molecular Dynamics, Sunnyvale, CA, USA).