2.3 Western blotting
After performing 12% SDS-polyacrylamide gel electrophoresis, protein
was then transferred onto a polyvinylidene difluoride (PVDF) membrane
(Bio-Rad, USA) with electroblotting at 35 volts for 16 hours in transfer
buffer. Non-specific protein was blocked by incubating the PVDF membrane
with TBST containing 3% (w/v) BSA (Sigma-Aldrich, USA) for 1 hour,
followed by washing with TBST three times. Membrane was then stained
with specific mouse anti-human IL-18 (R&D systems, USA) at 1:3,000
dilution for 1 hour and washed three times for 5 min each in TBST with
gentle agitation. Horse radish peroxidase-conjugated goat anti-mouse IgG
(R&D system, USA) was added at a dilution of 1:10,000 in TBST
containing 3% (w/v) BSA and incubated for 1 hour at room temperature.
After washing for three times in TBST, IL-18 were detected by Luminata
Forte Western HRP substrate (Millipore, USA) with UVITEC Cambridge Gel
Documentation System (UVITEC, UK).