Ab69617

Salmonella strains chromosomally producing 3×FLAG-tagged proteins were used for bacterial infection, and intracellular bacteria were isolated at the indicated times. Bacterial pellets were resuspended in the SDS loading buffer. After SDS-PAGE separation, bacterial proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Individual samples were probed with primary antibodies specific for FLAG (Sigma-Aldrich, F3165) (1:2,000) or Salmonella DnaK (Abcam, ab69617) (1:5,000) and horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma-Aldrich, A4416) (1:5,000).

ETEC strains H10407 and 1392/75-2a were grown to stationary phase in CFA^{50 (link),51 (link)} broth with or without decanoic acid in 0.4% vol/vol DMSO. Pilins were released from the outer membrane by incubating ETEC in 1/10 volume PBS at 65 °C for 20 min. Supernatants were clarified by centrifugation to remove insoluble material. Stationary phase whole cell lysates of ETEC or 5 μg pilin supernatants were subjected to SDS-PAGE then transferred to PVDF membranes or stained with 0.1% Coomassie R-250 in 40% ethanol, 10% acetic acid. PVDF membranes were blocked in TBS-Blotto (25 mM TrisCl pH 7.6, 150 mM NaCl, 5% wt/vol powdered nonfat milk). Antibodies against CexE homologs CexEα (H10407) and CexEε (1392/75-2a) were produced by immunization of rabbits (Proteintech Group, Inc.) with purified antigens and used at a dilution of 1:5000 in TBS-Blotto with 0.05% vol/vol Tween20^{45 (link),48 (link)}. The primary antibody against DnaK (AbCam ab69617) was used at a 1:10,000 dilution. HRP conjugated goat anti-rabbit (Santa Cruz Biotechnology sc-2030) and goat anti-mouse (Jackson ImmunoResearch 115-036-062) antibodies were used at 1:10,000 dilutions. Chemiluminescence and Coomassie staining was detected with an Odyssey FC Imaging System (LI-COR Biosciences).

HeLa cells were infected with Salmonella strains carrying pBR-GFP2 plasmid and pWSK29 carrying HA-tagged alleles of sopE2 or sopB. The infection procedure was performed as above with Salmonella cultures that were grown overnight under microaerobic conditions. Cell fixation was performed by incubation with 5 ml of 4% formaldehyde for 15 min under gentle shaking. Five ml of PBS +/+ were added and the cells were centrifuged for 5 min at 2,700 × g. Subsequently, the cells were washed three times with PBS +/+ and finally resuspended in PBS +/+ containing 1% BSA. The cells were stored at 4°C until sorting. 1×10⁶ GFP-positive cells were centrifuged for 5 min at 247 × g, and resuspended in 100 μl sample buffer. Western blot of the infected cells was performed as previously described [111 (link)] using the anti-2HA (Abcam ab18181; 1:1,000), anti-FliC (Abcam ab93713; 1:200,000) and anti-DnaK (Abcam ab69617; 1:10,000) antibodies.