Fig. S3.

ELISA and pull-down assay of the interactions between r-0583 and various mammalian sera, or rabbit and goat IgG. (A) ELISA was used to assess the ability of purified r-0583 to bind to serum components. Sera from healthy goats (i.e., goats that have never been in contact with any mycoplasma strains) were coated on an ELISA plate at 50 µg/mL in bicarbonate/carbonate 0.1 M pH 9.6 buffer. Binding to the coating was detected using a mouse anti-polyhistidine primary antibody and an anti-mouse AP-conjugated secondary antibody. Alkaline phosphatase activity was measured using pNPP substrate in diethanolamine buffer pH 9.8 at 37 °C. Error bars for the goat serum coating samples correspond to the SD from eight biological samples. Error bars for the BSA coating and no-coating samples correspond to the SD of three technical replicates each. (B) Purified r-0583 is bound to a Ni-NTA resin via its histidine tag, and incubated with normal serum from sheep, rabbit, horse, human, mouse, and bovine. After extensive washing, proteins bound to the resin are eluted with a pulse of imidazole and analyzed by SDS/PAGE. The control experiments include the bait protein r-0583 without serum (lane: r-0583) and the naked resin incubated with the serum (lane: serum). Black arrows indicate the heavy and light chains of immunoglobulins (55 kDa and 25 kDa, respectively). Identification of the proteins was confirmed by LC/MS-MS analysis. (C) ELISA was used to assess the ability of purified r-0583 to bind to IgG. Goat or rabbit purified IgG were coated on an ELISA plate at 50 µg/mL in bicarbonate/carbonate 0.1 M pH 9.6 buffer. Binding to the coating was detected using a mouse anti-polyhistidine primary antibody and an anti-mouse AP-conjugated secondary antibody. Alkaline phosphatase activity was measured using pNPP substrate in diethanolamine buffer pH 9.8 at 37 °C. Error bars correspond to the SD of six technical replicates.