Horseradish peroxidase conjugated goat anti mouse igg

The separated protein spots by 2-DE were transferred to a polyvinylidene fluoride (PVDF) membrane with a wet transfer cell (400 mA, 2.5 h). The PVDF membranes with the ES proteins were blocked with 5% skim milk in TBST (80 ml) for 1 h at room temperature. The TBST-blocked PVDF membrane was incubated (overnight, 4 °C) with T. pseudospiralis-infected swine pooled sera diluted 1:1000 in TBST. After completion of the incubation, the PVDF membrane was washed with the TBST solution (5 min × 3), and then the membrane was again incubated with the horseradish peroxidase-conjugated goat anti-mouse IgG (BioRad, Hercules, USA) (1:8000, 1 h, room temperature). The membrane was washed with TBST solution (5 min × 3) and visualized. Uninfected sera from the same pigs were used as a parallel negative-control. The negative-control experiment used the same method as mentioned above.
The scanned images of the Coomassie brilliant blue-stained 2-DE gels combined with the visualized western blot membranes were input to Image Master 2D Platinum 5.0 (GE) to identify species-specific spots.

Protocols for immunoblotting were as described previously (Inubushi et al., 2022 (link)). In brief, cells were lysed in ice-cold RIPA buffer containing 50 mmol/l Tris-HCl (pH 7.6), 150 mmol/l NaCl, 1% Nonidet P40 Substitute, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS). A protease inhibitor cocktail was purchased from Promega. Following a 30-min lysis period on ice, lysis samples were centrifuged at ∼20,000 g for 20 min at 4°C to prepare cell lysates. A total of 10 μg lysate was then subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on an 8-16% Tris-glycine gel (Invitrogen), followed by electroblotting onto an Immobilon PVDF membrane (EMD Millipore). ECL Western Blotting Substrate (07880; Nacalai Tesque) was used to detect signals. The following antibodies were used: anti-STAT3 (1:1000; 9139, Cell Signaling Technology), anti-pSTAT3 (1:1000; 9145, Cell Signaling Technology), anti-α-tubulin (1:1000; T6074, Sigma-Aldrich), horseradish peroxidase-conjugated goat anti-rabbit IgG (1:500; 1706565, Bio-Rad) and horseradish peroxidase-conjugated goat anti-mouse IgG (1:500; 1706515, Bio-Rad).