Secretion of SteA to the culture media.

<p>The presence of SteA-3xFLAG in cell lysates (from 10⁸ cells) and culture supernatants (from 2.5×10⁸ cells) was analyzed by immunoblotting with anti-FLAG antibodies as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026930#s4" target="_blank">Materials and Methods</a> section. The immunoblots were probed for GroEL as a loading control for the cell lysates and to test unspecific leakage of proteins into the supernatants. All the strains used produced a 3xFLAG version of SteA and were derivatives of the wild-type strain 14028 (wt), a <i>prgH</i> mutant (lacking the T3SS1) and an <i>ssaV</i> mutant (lacking the T3SS2). Bacteria were grown at 37°C using the following conditions: (A) SPI-1 conditions (LB with 0.3 M NaCl, overnight without shaking); (B) SPI-2 conditions (LPM pH 5.8, 24 h with shaking); (C) SPI-2 conditions followed by shifting to pH 7.2 and 1 h incubation; (D) SPI-2 conditions in an <i>ssaL</i> background.