Mouse α dnak

Protein fractions were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane, and blocked in Tris-buffered saline with 0.1% (w/v) Tween (TBST) containing 5% skim milk. Membranes were then probed using the following primary antibodies: mouse monoclonal anti-Tir (1:2000) for full-length and C-terminal fragments, rat polyclonal anti-Tir from C. rodentium (1:2000) [49 (link)] for N-terminal fragments, rat polyclonal anti-NleA from EHEC (1:2000) [50 (link)], mouse α-DnaK (Stressgen, 1:5000), mouse α-FLAG M2 (Sigma, 1:5000), mouse α-His6 (GE Healthcare, 1:3000), or goat α-GAPDH (R&D Systems Inc., 1:5000). The blots were then developed using the following secondary antibodies: goat anti-mouse (1:5000, Jackson), goat anti-rat (1:2000, EMD Millipore), or donkey α-goat (Santa Cruz Biotechnology, 1:5000) conjugated to horseradish peroxidase, and imaged using the Clarity Western ECL (BioRad) or SuperSignal West Femto Maximum Sensitivity (ThermoFisher) substrates and a ChemiDoc XRS+ (BioRad).

S. sonnei cultures were induced with 1 mM IPTG at OD₆₀₀=0.2. After a further 60 min of growth, liquid cultures were harvested and concentrated to an OD₆₀₀=5.0. The cell pellet (cellular fraction) was resuspended in reducing Laemmli buffer and heated at 95 °C for 5 min. The supernatant fraction was filter sterilised with a 0.22 µm filter and precipitated with trichloroacetic acid. The precipitated supernatant faction was resuspended in reducing Laemmli buffer and heated at 95 °C for 5 min. All samples were separated on a 15 % SDS-PAGE and proteins transferred to PVDF membranes. Membranes were probed with mouse α-HA (Biolegend) or mouse α-DnaK (Stressgen). Followed by an anti-mouse HRP conjugated secondary antibody (Sigma Aldrich). Membranes were then incubated with ECL reagent and visualised on a BioRad ChemiDoc XRS+system.