A mixed culture of EPEC and V. cholerae shows elevated T3SS activity compared to an EPEC pure culture. Pure overnight cultures of EPEC, V. cholerae, and E. coli DH10B were sub-cultured into fresh 1:1 (v/v) DMEM:plain LB mixture as pure or mixed cultures. The cultures were grown under semi T3SS-inducing conditions for 6 h, and then bacterial pellets and supernatant (sup) were separated and analyzed. The secreted proteins were concentrated from supernatants of bacterial cultures and analyzed by 12% SDS-PAGE and western blot analysis using anti-EspB and anti-EspA antibodies for the secretion of the EspA and EspB translocator proteins. The expression of the structural T3SS protein, EscJ, and the effector protein, Tir, which should remain mostly within the bacterial cytoplasm at this stage, were analyzed by subjecting the bacterial pellets to SDS-PAGE and western blot analysis using an anti-EscJ and anti-Tir antibodies. Samples were also probed with anti-DnaK to demonstrate equal loading.