Mouse anti ha 11

Samples were subjected to SDS-PAGE and transferred to nitrocellulose (pore size: 0.45 μm; Amersham Protran) or PVDF (pore size: 0.45 μm; Amersham Hybond) membranes. The blots were then 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, unless otherwise indicated), 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-HA (Abcam Inc.), diluted 1:1,000; mouse anti-HA.11 (Covance), diluted 1:1,000; mouse anti-V5 (Invitrogen), diluted 1:1,000; mouse anti His (Pierce), diluted 1:2000; mouse anti-JNK (BD Pharmingen), diluted 1:1,000 in Tris-buffered saline (TBS); and mouse anti-actin (MPBio), diluted 1:10,000. Antibodies directed against T3SS components included mouse anti-EspA, mouse anti-EspB and mouse anti-Tir, all a generous gift from Prof. B. Brett Finlay (University of British Columbia, Canada). Horseradish peroxidase-conjugated (HRP)-goat anti-mouse (Abcam Inc.), diluted 1:10,000, was used as the secondary antibody. Representative western blots of at least three independent experiments are presented in the results section.

For immunocytochemistry, all steps were performed at RT. HEK-293 cells grown on poly-L-lysine–coated coverslips were fixated for 10 min with 4% paraformaldehyde in 0.2 M phosphate buffer. After three washing steps in PBS, cells were incubated with the blocking solution (2% bovine serum albumin and 10% goat serum in PBS) for 30 min. The primary antibody solution (mouse anti-HA.11; 1:1000; Covance) was added in the carrier solution (0.3% Triton X-100, 1% bovine serum albumin, 1% goat serum in PBS) for 1 h. After washing three times in PBS, the secondary antibody, which was conjugated to a fluorescent probe (Alexa Fluor 488 goat anti-mouse; 1:1000; Thermo Fisher), was incubated for 1 h. After three washes in PBS, cells were mounted onto glass slides with Mowiol (Roth) and 4′,6-diamidin-2-phenylindol (1:1000; Roth). Photomicrographs were taken using a BioZero fluorescence microscope (Keyence).