Hrp conjugated goat anti rat

Samples were subjected to SDS-PAGE and transferred to nitrocellulose membrane (pore size, 0.45 µm; Bio-Rad). The membranes were blocked for 1 h with 5% (w/v) skim milk/PBST (0.1% Tween in phosphate-buffered saline), incubated with the primary antibody (diluted in 5% skim milk/PBST) for 1 h at room temperature or overnight at 4°C, washed, and then incubated with the secondary antibody (diluted in 5% skim milk/PBST) for 1 h at room temperature. Chemiluminescence was detected with EZ-ECL reagents (Biological Industries). The following primary antibodies were used: mouse anti-His (Pierce), diluted 1:2,000; rabbit anti-MBP (Thermo Fisher Scientific), diluted 1:1,000; mouse anti-DnaK (Abcam, Inc.), diluted 1:5,000; and mouse anti-actin (MPBio), diluted 1:10,000. Antibodies directed against T3SS components were a generous gift from Prof. B. Brett Finlay (University of British Columbia, Canada) and Prof. Rebekeh DeVinney (University of Calgary, Canada) and included mouse anti-Tir, rat anti-Intimin, and rat anti-Tir. Horseradish peroxidase-conjugated (HRP)-goat anti-mouse (Abcam Inc.), HRP-conjugated goat anti-rabbit (Abcam Inc.), and HRP-conjugated goat anti-rat (Jackson ImmunoResearch), diluted 1:10,000, were used as the secondary antibodies. Representative western blots of at least three independent experiments are presented in the Results section.