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The pET-28a expression system (Novagen) was used to produce C-terminally His-tagged RpoN within host E. coli BL21 (DE3) cells. A fragment of rpoN was amplified by PCR using the pET28a-rpoN-F/R primers (Supplementary Table 2). The PCR product was digested with NdeI and HindIII and ligated into the protein expression vector pET28a, which had been digested with the same enzymes. The resulting plasmid (pET28a-RpoN) was introduced into the E. coli BL21 (DE3) strain. An overnight culture of BL21 (DE3) harbouring the expression plasmid was used to inoculate LB medium containing the appropriate antibiotics. This cell culture was incubated with shaking at 37 °C until the OD₆₀₀ was 0.6–0.9, at which point production of His-tagged RpoN was induced by the addition of IPTG to a final concentration of 0.1 mM. His-tagged RpoN was purified using a Ni-NTA Fast Start Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. The purity of the protein was as high as 90%, as judged by SDS-Tris-glycine PAGE. His-tagged RpoN was quantitated using the Bio-Rad protein assay reagent kit (Quick Start™ Bradford, Bio-Rad) and stored at −80 °C for further use.

HEK293T cells were transfected with 24 μg flag-tagged DNA encoding CaMKII alpha (GenBank Accession No. AAH40457.1, OriGene Technologies) using Lipofectamine 2000 (Invitrogen) and incubated for 6 h. Culture medium was changed to fresh medium with or without 0.1 mM CaCl₂ and cells were incubated for 16 h. Cells were lysed in mammalian cell lysis CelLyticM (Sigma) including fresh Protease Inhibitor Cocktails. Cell lysates were centrifuged and the supernatant was incubated with ANTI-FLAG M2 Affinity Gel (Sigma) overnight at 4°C. Flag-tagged CaMKII was eluted with FLAG peptide (Sigma) after stringent washes. The FLAG peptide was removed by Amicon filter of 10-kDa (Millipore). Protein concentration was measured by Quick Start Bradford (Bio-Rad) and protein purity was checked by SDS-PAGE with Coomassie staining.

Third-stage larvae (L3) of A. simplex (s.l.) were collected from a silver scabbard fish (Lepidopus caudatus) fished in the Mediterranean Sea and L3 of Pseudoterranova sp. were collected from Atlantic cods (Gadus morhua) fished in the North Atlantic Ocean. L3 were extensively washed one by one in 2% acetic acid-phosphate buffered saline (PBS) and then each larva was cut into two parts, one part was kept in 90% ethanol for the molecular identification by PCR/RLPF [18 (link)], and the other part was frozen at -20 °C. L3 from the silver scabbard fish were identified as A. pegreffii, whereas L3 from the Atlantic cods were identified as P. decipiens. Crude worm extracts (CWE) were prepared from frozen L3. Briefly, 50 frozen L3 were suspended in 0.5 ml of PBS and then homogenized 5 times for 30 s and sonicated in ice 5 times for 60 s. The suspension was left overnight at 4 °C under magnetic stirring and further centrifuged at 1500× g and at 4 °C for 15 min. The protein content of the centrifuged supernatant was measured by the Bradford method (Quick Start™ Bradford, BIO-RAD, Hercules, CA, USA). Moreover, 140 and 80 additional live L3 assumed to be A. pegreffii and P. decipiens, respectively, on the basis of their morphology [19 ], host and geographical origins, were collected to be injected into BALB/c mice.

Protein extraction from formalin-fixed paraffin-embedded tissues was performed according to Guo and co-workers [47 (link)]. Briefly, paraffin blocks were cut (approximately 10 µm), deparaffined in xylene and rehydrated in decrescent graded alcohol (100, 90, 70, 50, 30% v/v). Samples were resuspended in 20 mM Tris pH 9 and 2% SDS and boiled for 20 min at 100 °C and 2 h at 80 °C. After centrifugation, supernatants were collected, and protein content quantified by Quick Start Bradford (Bio-Rad, Hercules, CA, USA). Fifty μg of proteins from each sample were diluted in Laemmli buffer and boiled for 5 min. Samples were subsequently resolved on SDS-PAGE and transferred to PVDF membranes. Membranes were blocked for 1 h at RT with 5% non-fat dry milk in TBS buffer (20 mM Tris and 105 mM NaCl) containing 0.1% Tween-20 (block solution) and then incubated overnight with primary antibody dissolved in block solution. The day after, membranes were washed three times in TBS containing 0.1% Tween-20 (TBST) and incubated in secondary anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA, USA) diluted 1:10,000 in block solution. Immunostained bands were detected by a chemiluminescent method (Clarity Western ECL Substrate, #1705061, Bio--ad, Hercules, CA, USA). Primary antibodies (α-SMA and tubulin) were diluted 1:1000 in block solution.

Nuclear extract of each hippocampus and frontal cortex was prepared as previously described [26 (link)]. Briefly, tissues were homogenized using a Dounce homogenizer in cold PBS supplemented with 0.5 mM PMSF, 2.5 μg/ml leupeptin and 2.5 μg/ml antipain, and centrifuged at 4°C for 30 sec at 12,000g. The supernatants (cytoplasmic extract) were reserved for immunoblot, and the pellets were resuspended in lysis buffer (1.5 mM MgCl₂, 10 mM KCl, 0.1 mM EDTA, 0.5 mM PMSF, 2.5 μg/mL leupeptin, 2.5 μg/ml antipain and 10 mM HEPES; pH 7.9) and incubated on ice for 10 min. After addition of NP-40 (10%), samples were vigorously mixed and centrifuged for 30 sec at 12,000g. Supernatant was discarded, and the pellet was resuspended in extraction buffer (25% glycerol, 1.5 mM MgCl₂, 300 mM NaCl, 0.25 mM EDTA, 0.5 mM PMSF, 2.5 μg/ml leupeptin, 2.5 μg/ml antipain and 20 mM HEPES; pH 7.9), incubated for 20 min on ice, and centrifuged for 20 min at 12,000xg at 4°C. The resulting supernatants containing nuclear proteins were stored at -80°C. Protein concentration was determined using the Bio-Rad (Quick Start Bradford,Richmond, USA) colorimetric assay [27 (link)].