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YscI is a secreted substrate of the T3SS. Wild-type (WT) and yscS and yscI mutant strains were grown at 37°C in BHI-minus-calcium medium. Secreted supernatant (SN) and cell pellet (P) fractions were separated by centrifugation, and proteins in the supernatant were TCA precipitated. Equal percentages of the two fractions were run on SDS-PAGE gels, and YscI was detected by Western blotting with a polyclonal antiserum.
