SDS-PAGE of secreted proteins (A, C) and Western blot for detection of SspA and Hla (B), in culture supernatant of USA300 after growth for 18–24 h in the presence of C16 (A) or C18 (C) fatty acids.

Cultures were grown with the indicated amounts of C16∶1▵6 (sapienic acid), C16∶1▵9 (palmitoleic acid), C16∶0 (palmitic acid), C18∶2 (linoleic acid), C18∶1 (oleic acid), C18∶3 (linolenic acid) or C18∶0 (stearic acid) fatty acids. Proteins in the cell-free culture supernatant were precipitated in ice-cold TCA, and after solubilization in SDS-PAGE reducing buffer, protein equivalent to 2.0 OD600 units of culture supernatant was loaded in each lane (A and C). For Western blot (C), 0.02 OD600 units of cell free culture supernatant were subjected directly to SDS-PAGE, prior to detection with specific antisera (see Materials and Methods).