Bacterial cultures of S. aureus USA300 or esxD mutants harboring or not a complementing plasmid (pesxD) were grown to OD600nm of 1.0 and separated into cell and medium fractions. Proteins were precipitated with trichloroacetic acid, separated by SDS-PAGE and detected by immunoblotting with specific antibodies (α-EsxA, α-EsxB, α-EsxC, α-EsxD, α-EssB, α-EssD, and α-Hla or α-L6 for fractionation and loading controls).