Binding of M. hyopneumoniae EF-Tu to Fibronectin

To explore if M. hyopneumoniae EF-Tu could bind to fibronectin, the protein-protein interactions method far-Western blot (far-WB) was performed. Total 20 μg recombinant proteins, including rEF-Tu and a negative control MRP-D2, which was previously verified not to bind to fibronectin (Li et al., 2017 (link)) were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Li et al., 2015 (link)). After blocked with 5% (w/v) skimmed milk, the membrane was incubated with 5 μg/mL fibroenctin (Sigma), followed by incubation with rabbit anti-fibronectin antibody (Boster; 1 μg/mL) as the primary antibody, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Boster; 1:5,000 dilution) as the secondary antibody. Finally, the membrane was developed with ECL substrate using a ChemiDoc XRS+ system. Polyclonal antibody against rEF-Tu was used as the positive control with the same process.