Figure 7. STM1485 is required for the SPI-2 translocon formation. Surface translocation of SPI-2 encoded translocon proteins (SseB and SseD) in the ΔSTM1485 (A). WT and ΔSTM1485 were grown in ISM media at pH 5.0 and then cell pellet, surface and supernatant fractions were isolated as described in Materials and Methods. By immunoblotting, the presence of SseB protein in all three fractions was analyzed. Secretion of SPI-2 effector protein (B). STM-WT, ΔSTM1485 and ΔssaV mutant strains carrying the His-tagged SseJ construct were grown under SPI-2 inducing conditions (F-media, pH 5.0) for overnight and secreted proteins were precipitated from the culture supernatant. We evaluated the cell pellet and supernatant fractions for intracellular and secreted levels of the SseJ-His by immunoblotting. Detection of SseD in vitro and in intracellular bacteria (C and D). WT-GFP and ΔSTM1485-GFP were grown in ISM media at pH 5.0 for overnight. Bacteria were harvested and preceded for immunostaining (C). RAW264.7 cells were infected with WT-GFP and ΔSTM1485-GFP. Twelve hours post-infection cells were fixed and immunostained (D). Samples were analyzed using a confocal laser scanning microscope (LSM Meta, Zeiss). Note the appearance of SseD (red) on the surface of intracellular bacteria (green) (*) and also distant to the bacteria (arrow). Scale bar represent the 2 µm.