Typhimurium expressing SscA-FLAG or SseC-FLAG from the IPTG-inducible pFLAG-CTC plasmid. A strain carrying empty plasmid YH25448 was used as a control. Strains were grown overnight in Luria-Bertani broth (LB) and sub-cultured 1:50 into 50 ml of LPM medium and grown to an optical density (OD600) of 0.6 at 37°C. Cultures were then centrifuged at 3000 × g for 10 min, and re-suspended
in phosphate buffered saline (PBS) containing mini-EDTA protease inhibitor cocktail (PBS-PI; Roche). Cells were lysed by 6 pulses of sonication for 30 sec each, with 60 sec intervals between sonication (Misonix Sonicator 3000, Misonix). Lysates were centrifuged at 3000 × g for 15 min at 4°C and the supernatant removed to obtain check details the cytosolic protein fraction. M2-agarose beads conjugated with anti-FLAG antibodies (F-gel; Sigma) was equilibrated with PBS-PI containing 10 μg/ml bovine serum albumin (BSA) for 60 min at 4°C with rocking and washed with PBS-PI three times. The beads were mixed with the cytosolic protein fractions and incubated for 16 h at 4°C with end-on-end mixing. Unbound proteins were removed by centrifuging the F-gel at 1000 ×
g for 5 min and removing the supernatant. The F-gel was washed ten times with PBS-PI containing 0.1% Triton-X100 before eluting bound proteins into sodium dodecyl sulfate (SDS)-sample buffer (1M Tris pH 8.0, 20% SDS, 0.5 M EDTA pH 8, 10% glycerol, 200 mM dithiothreitol). Bound proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad). Western blots were probed with antibodies to SseC (a gift from Dr. Michael Hensel), the FLAG epitope (Sigma), or the His6 tag (Qiagen). For reciprocal Megestrol Acetate co-immunoprecipitations,
a strain containing a plasmid encoding sscA-HIS 6 and a second compatible plasmid encoding sseC-FLAG was used. SscA-His6 was induced with arabinose and SseC-FLAG was induced with IPTG as above. In this experiment, the anti-FLAG gel was used for JNK-IN-8 order immunoprecipitations and anti-His antibody used in immunoblotting as described above. Protein secretion assasy Wild type S. Typhimurium and ΔsscA strains were grown overnight in LB and sub-cultured 1:50 into LPM and grown to OD600 of 0.6. Cultures were then centrifuged for 2 min at 10,000 × g and the supernatant was filtered through a 0.2 μm filter (Pall Scientific) and precipitated with 10% trichloroacetic acid (TCA). Precipitated secreted proteins were centrifuged at 16,000 × g at 4°C for 30 min and the pellets were washed with acetone and dissolved in SDS-sample buffer. The whole cell lysate fraction was made by dissolving the bacterial pellet in SDS-sample buffer.