Evaluation of the presence and stability of the NadA4 protein. Unless indicated otherwise, all samples were loaded on the gel after addition of sample buffer and boiling for 10 min. Lane 1, strain 2996 (NadA3) total protein; lane 2, strain 65/96 (NadA4) total protein;lanes 3, 4, and 5, strain 65/96 (NadA4) OMVs boiled for 30 min, resuspended in 2% Triton X-100, and resuspended in 2% SDS, respectively; lanes 6, 7, and 8, strain 2996 (NadA3) OMVs boiled for 30 min, resuspended in 2% Triton X-100, and resuspended in 2% SDS, respectively; lane 9, strain NGH38 OMVs (negative control).