FIG. 5.

Xylene extraction of yersiniae. Y. enterocolitica W22703 was grown at 37°C and induced for type III secretion by the chelation of calcium ions. Culture aliquots were centrifuged, and the bacterial sediment was washed and suspended in TSB. After extraction with xylene and centrifugation, the extract supernatant (S) and pellet (P) were separated, precipitated with acetone, and analyzed by immunoblotting. Small amounts of YopD, YopE, and LcrV, known type III secretion substrates, were extracted with xylene. The cytoplasmic protein SycE, the cytoplasmic membrane protein LcrD, and the outer membrane protein YscC were used as controls. YopN, another type III secretion substrate, Gst-TyeA, and TyeA were not released from yersiniae by treatment with xylene.