2.7 | Immunoblotting
Samples were subjected to SDS-PAGE and transferred to nitrocellulose
membranes (pore size: 0.45 μm, Bio-Rad) or polyvinylidene difluoride
(PVDF, Mercury, Millipore). The blots were blocked for 1 hr 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
hr at room temperature or overnight at 4°C), washed, and then incubated
with the secondary antibody (diluted in 5% skim milk-PBST, for 1 hr at
room temperature). Chemi-luminescence was detected with EZ-ECL reagents
(Biological Industries). The following primary antibodies were used:
mAb-EspB-B7, diluted 1:1000; mouse anti-EspB (a gift from Prof. Finlay,
University of British Columbia), diluted 1:1000; mouse anti-His
(Pierce), diluted 1:2000; mouse anti-JNK (BD Pharmingen), diluted 1:1000
in TBS; and mouse anti-actin (MPBio), diluted 1:10,000. The following
secondary antibodies were used: horseradish peroxidase-conjugated
(HRP)-goat anti-mouse (Abcam Inc.) and HRP-conjugated goat anti-human
(Abcam Inc) antibodies.