2.11 | Effector translocation activity
Translocation assays were performed as previously described (Baruch et
al., 2011). Briefly, HeLa cells (8 × 105 cells per
well) were infected for 3 hr with EPEC strains that were pre-induced for
3 hr for T3SS activity (pre-heated DMEM, statically, in a
CO2 tissue culture incubator). Cells were then washed
with PBS, collected, and lysed with RIPA buffer. Samples were
centrifuged at 18000 × g for 5 min to remove non-lysed cells, and
supernatants were collected, mixed with SDS-PAGE sample buffer, and
subjected to western blot analysis with anti-JNK and anti-actin
antibodies (loading control). Uninfected samples and the ΔescNmutant strain-infected samples were used as negative controls. To
evaluate the ability of mAb-EspB-B7 to inhibit EPEC translocation
activity, 400 nM of mAb-EspB-B7 were added to a sample infected with WT
EPEC.