Bovine serum albumin (bsa)

Human recombinant AMH and GH, and mouse recombinant IGF1, were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Forskolin and the other analytical reagents were purchased from Merck (Milan, Italy). GH and IGF1 were reconstituted in PBS, while AMH in 4 mM HCl with 0.1% bovine serum albumin (Euroclone, Milan, Italy), according to manufacturer’s instructions.

Cell pellets were resuspended and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol), complemented with protease and phosphatase inhibitors cocktail (ThermoScientific). Total proteins extracts concentrations were determined through Bradford assay (Bio-Rad). Cytosol and nucleus protein fractions were obtained as previously described [47 (link)].
After 1 h blocking with 5% non-fat dried milk (EuroClone) or bovine serum albumin (SERVA) in Tris-buffered saline with 0.1% Tween (TBS-T), membranes were incubated with primary antibodies at 4 °C overnight. Primary antibodies used: anti-pFGFR1 (06-1433, Millipore), anti-FGFR1 (Abcam ab137765), anti-pSTAT3 (D3A7, Cell Signaling), anti-STAT3 (06596, Millipore), anti-pAKT1 (ab81283; Abcam), anti-AKT1 (ab32505; Abcam), anti-pERK1/2 (ab32538; Abcam), anti-ERK1/2 (ab17942; Abcam), anti t-GFP (TA150041, Origene) and β-actin (Sigma, A5441). After membrane incubation with horseradish-peroxidase-conjugated anti-rabbit secondary antibody (Immuno Reagents), the positive bands were visualized using the ECL kit SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) as previously shown [48 (link)].

Immunoassays were performed as previously standardized and taking into account the direct ELISA results [24 ]. Plates were coated with 100 μL/well of T. gondii crude extract from RH strain tachyzoites (2 μg/mL) in carbonate buffer at 4 °C overnight and washed as described above; next, non-specific binding sites were blocked with 200 μL of 1% bovine serum albumin (Euro-Clone, Milan, Italy, product JI001000173) for 30 min at 37 °C. Wells were washed and 100 μL of each serum sample, diluted 1:100, were added and incubated for 2 h at 37 °C. One hundred μL/well of Protein A (Sigma-Aldrich Corp.) diluted 1:250 were added and incubated at 37 °C for 2 h. After performing the appropriate washes, the antigen-antibody complexes were revealed and read as described above for direct ELISAs.
Because there were no wild rodent positive or negative control sera for detection of T. gondii infection, the cut-off point was calculated using the frequency distribution of the absorbance values, which had normal distribution, using their average plus 3 times the standard deviation, as previously described [23 (link)].