Immunoblot and immunohistochemical analyses.

Antibodies were produced against the open reading frames (ORFs) of EgMKK1 and EgMKK2, which were amplified using primer pairs F20/R20 and F21/R21 (see Table S1 in the supplemental material), subcloned into the pET-28α(+) expression vector, and expressed in Escherichia coli. Antisera were produced in rabbit after immunization against the recombinant protein under conditions previously described (6).

Total protein extracts were obtained by homogenizing E. granulosus sensu stricto PSCs or cysts in lysis buffer (Life Technologies, USA). Analyses of the parasite and recombinant proteins were performed as previously described (6). The following primary antibodies were used to detect total EgMKK1 and EgMKK2: anti-EgMKK1 (1:2,000), anti-EgMKK2 (1:2,000), or preimmune rabbit serum (1:2,000), overnight at 4°C. Alkaline phosphatase-conjugated goat secondary antibody was used as the secondary antibody (1:1,000) (Cell Signaling Technology, USA). Immunoreacting bands were visualized using the alkaline phosphatase substrate kit (Thermo Fisher Scientific, USA).

To investigate the immunolocalization of EgMKK1 and EgMKK2, fresh PSCs and cysts were generated as previously described (6). The sections were fixed for 15 min and then blocked in blocking buffer and incubated with rabbit polyclonal antibody (EgMKK1 at 1:1,000 and EgMKK2 at 1:2,000) overnight at 4°C. At the end of the incubation, samples were washed with phosphate-buffered saline (PBS) and reacted with Alexa 488 fluorochrome-conjugated F(ab′)² antibody (Cell Signaling Technology, USA) for 2 h at room temperature in darkness. The nucleus was stained with 4ʹ,6-diamidino-2-phenylindole (DAPI; Cell Signaling Technology, USA) for 5 min. The sections were imaged with a confocal laser scanning fluorescence microscope (Leica, Wetzlar, Germany). Negative controls were prepared with preimmune rabbit serum.