Western blot validation of comparative proteomics analysis

Equal amounts (40 μg) of each protein sample were separated on a 12% SDS-PAGE gel, and proteins were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Germany) and developed with Ponceau-S as the loading control. After blocking with TBST buffer comprising 20 mM TRIS–HCl (pH 7.6), 150 mM NaCl and 0.1% Tween-20 containing 5% skimmed milk, membranes were probed with anti-rFBA antibody (1:2000 dilution). Horseradish peroxidase (HRP)-conjugated secondary antibody (1:10 000 dilution) was used for final identification. ImageJ software was used to calculate the optical density (OD) of the corresponding bands. The OD of FBA from different samples was normalised to that of the Ponceau-S-stained membrane. The abundance of FBA in M. hyopneumoniae strain 168L is expressed as the percentage of that in M. hyopneumoniae strain 168. Three replicates were subjected to statistical analysis by SPSS 20.0.