Translocated SseF is an integral membrane protein.

<p>A) Schematic representation of the procedure used for the analysis. HeLa cells were infected at an MOI of 10 with the <i>sseF</i> strain harboring a plasmid for the expression of <i>sseF</i>::HA (WT SseF) or <i>sseF</i>_Δ200–205 (Δ200–205) as indicated. Cells were harvested 16 h p.i. and subjected to subcellular fractionation after mechanical lysis. The membrane fraction was used for extraction under conditions of different stringency. The cytoplasmic fraction (C) was separated from the membrane pellet as described in the Experimental Procedures. B) The membrane fraction was incubated with high salt/high pH buffer (K) or with alkaline buffer (N) in order to extract peripheral membrane proteins with hydrophobic or hydrophilic interaction with membranes, respectively. C) The membrane fraction was treated with Triton X-100 buffer (T) to extract integral membrane proteins, and with alkaline buffer (N) as control. C indicates the cytosolic fraction, M the membrane extract and I the insoluble membrane pellet. The various fractions were subjected to SDS-PAGE on 12% gels and the proteins were transferred onto nitrocellulose membranes. The HA epitope tag was detected using antibodies against the HA-tag and as controls, the Western blots were incubated with antibodies against LAMP1 as an integral protein of endosomal membranes.