BMDCs were resuspended 330 μL of PBS containing 3 μL of a protease inhibitor cocktail (Halt™ protease inhibitor-single-use cocktail; Thermo Scientific, Rockford, IL, USA) and 33 μL of Triton-X-114, and the mixture was alternatively vortexed and chilled for 5 min before being sedimented (10 000 × g for 10 min at 4°C) to remove the insoluble residue. The extraction was repeated twice. The pooled supernatants were incubated for 5 min at 37°C to allow the formation of micelles. Water phase and Triton phase containing the membrane proteins were separated after sedimentation at 10 000 × g for 5 min at room temperature. The water phase was removed. Membrane proteins in the Triton

phase were washed with 1 mL of PBS plus protease inhibitor cocktail (1%) and precipitated as described (22). The pellets were dried, resuspended in sample buffer for Selleck C59 wnt SDS–PAGE according to Laemmli (23) and stored at −20°C until use. Membrane protein fractions prepared as described above were heated at 65°C for 20 min before loading on a 12% gel. SDS–PAGE was performed as previously described (23), and the proteins were electrophoretically transferred onto a nitrocellulose sheet (Schleicher & Schüll, BA85). Blots were rinsed in PBS and incubated in blocking buffer, including PBS-Tween 20 (0·3%) plus 5% fat-free milk powder, during 2 h at room temperature.

The nitrocellulose membrane was then washed three times and incubated RAD001 chemical structure with the first antibody, rat anti-mouse MHC class II (I-A/I-E) (eBioscience,

THP, Vienna, Austria) diluted (1 : 500) in PBS-Tween 20 (0·3%) during 1 h. The membrane was subsequently washed three times and incubated with the secondary antibody, anti-rat-IgG-alkaline phosphatase-conjugate (Sigma Chem. Co.) for 1 h at a dilution (1 : 1000) in PBS-Tween 20 (0·3%). To visualize bands, the membrane was incubated in 10 mL of substrate buffer [MgCl2 (10 mm) + NaCl (100 mm) + Tris (100 mm)] adjusted to pH 9·5 and supplemented with 66 μL BCIP and 66 μL NBT, prepared, respectively, in 100% and 70% of dimethyl ASK1 formamide (DMF). At the last step, the membrane was transferred into water to stop the enzymatic reactions and then dried on absorbent paper at room temperature. The influence of peritoneal DCs isolated from metacestode-infected mice and naïve mice on the activation of naïve CD4+ pe-T cells was studied using an assay described previously (24); 5 × 104 of naive CD4+pe-T cells were stimulated with Con A (2 μg/mL) in the presence of varying numbers of naive pe-DCs or AE-pe-DCs ranging from 5 × 103 to 5 × 104 cells. Cells were incubated in 200 μL total volume of complete medium (RPMI-1640 supplemented with 10% FCS (v/v), 2 mm l-glutamine, 1 mm sodium pyruvate, 1 mm nonessential amino acids, 0·05 mm mercaptoethanol, 100 U/mL penicillin per streptomycin) during 48 h at 37°C and 5% CO2. Cell proliferation was assayed using the colorimetric BrdU (5-bromo-2-deoxyuridine) cell proliferation ELISA kit (Calbiochem, Merck Chemicals, Switzerland).