Cytokine and antibody arrays
Human Inflammation Antibody Array C3 (RayBiotech) was used to detect
cytokines and factors in conditioned media from cells under indicated
conditions. Cells were plated in 6-well and treated with IDX, cis or
combination of both. 72 h later, culture medium was centrifuged prior to
assay on the human cytokine array according to the manufacturer’s
guidelines.
Western
blot
Cells were lysed in RIPA buffer containing protease and phosphatase
inhibitors on ice for 45 min and collected by centrifuging at 150,000
rpm for 15 min at 4 oC. Protein concentrations were
measured by the bicinchoninic acid protein assay kit (Pierce). Equal
amounts of protein were solubilized in laemmili buffer, boiled for 5 min
and then separated by 10 % SDS-PAGE before transferring them onto
nitrocellulose membranes. Membranes were blocked and probed with
specific primary antibodies (Supplementary Table 2), detected by using
appropriate HRP-conjugated secondary antibodies and visualized by
incubation with enhanced chemiluminescence reagent (ECL, GE Healthcare)
and exposure to film.