Goat anti mouse hrp

Overnight bacterial cultures were 1:100 transferred in DMEM until the culture reached an OD₆₀₀ of 1.0. The bacteria were collected and washed three times with PBS, and then the cells were lysed by ultrasound. Equal amounts of total proteins (40 μg) were loaded on a 12% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The blot membrane was blocked with QuickBlocK^TM blocking buffer (Beyotime, Shanghai, China) for 30 min at room temperature, followed by incubation with polyclonal antisera (mouse) against Tir or intimin and with primary antibody (1:10,000 dilutions for anti-DnaK antibody; Abcam, Cambridge, UK) for 2 h and another 1 h of incubation with secondary antibody (1:5000 dilutions for the goat anti-mouse-HRP; CWBIO; Beijing, China). Blots were detected using the ECL-enhanced chemiluminescence reagent (CWBio, Beijing, China), and protein levels were quantified using ImageJ software (NIH, Version: 1.8.0). Three independent biological replicates were performed for each experiment.

To analyse HilA protein levels in wild-type, ΔloiA, ΔhilD, ΔloiAΔhilD, ΔloiAΔhilD+pHilD and ΔloiAΔhilD+pLoiA strains, the corresponding 3×FLAG-tagged strains were grown in SPI-1-inducing conditions and then collected. To analyse the influence of osmolarity and O₂ on the production of LoiA protein, the 3×FLAG-tagged strain (WT loiA-FLAG) was grown under the conditions indicated as described above (high O₂ or low O₂; high salt or low salt). Bacteria were pelleted by centrifugation, washed with PBS, resuspended in 100 μl SDS-polyacrylamide gel electrophoresis solubilization buffer (normalized for OD₆₀₀ to ensure equivalent bacterial numbers) and lysed at 100°C for 10 min. Proteins were separated via 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were treated with 5% nonfat milk for 1 h to block non-specific binding and incubated with primary antibody raised in mice against the FLAG-tag (1:2,500 dilution, Sigma) or DnaK (1:5,000 dilution, Abcam) for 1 h, followed by washing in TBST. The blots were further incubated with the secondary antibody goat anti-mouse-HRP (1:5,000 dilution, CWBIO) for 1 h. Blots were washed in TBST followed by detection with the ECL enhanced chemiluminescence reagent.