The MbovP280 of mycoplasma bovis immunoprecipitates with the wole cell lysates of bovine macrophage (BoMac),and then LC-MS/MS was used to identify the MbovP280-binding ligands

An IP–MS method was used to screen for MbovP280-binding proteins. Briefly, BoMac cells were cultured, harvested, and lysed in RIPA buffer supplemented with cOmplete Protease Inhibitor Cocktail (Roche, Mannheim, Germany). The whole-cell lysates (400 μg) were incubated with 10 μg of either rMbovP280 or rMbovP280∆210–269 for 1 h at 4 C. Mouse antiserum (5 μg) directed against MbovP280 was added to the lysates for 12 h at 4 C, and then 50 μl of Protein A/G Agarose Beads (Beyotime) was added for an additional 1 h. The immunoprecipitates were extensively washed with NP-40 buffer and eluted with SDS loading buffer by boiling them for 5 min. The cellular proteins that coprecipitated with rMbovP280 or rMbovP280∆210–269 were resolved with SDS-PAGE and stained with silver staining.