Concentrations of TNF-α and IL-8 were measured in cell culture supernatants by enzyme linked immuno-absorbent assays (ELISA). Specific kits for porcine TNF-α and IL-8 (R&D Systems, Minneapolis, MN, USA) were used according to the manufactured instructions as already described20 (link), 66 (link).
The expression of phosphorylated p38 MAPK in IPEC J2 cell lysates was analyzed by western blot as already described26 (link). Briefly, total proteins were separated on SDS-PAGE, transferred onto nitrocellulose membranes, incubated with Rabbit anti-phospho-p38 (Cell Signaling Technology, Danvers, MA) overnight and further incubated with CFTM770 goat anti-rabbit IgG (Biotium, Hayward, CA). Mouse anti-β-actin (Cell Signaling Technology) was used as control. Membranes were analyzed using an Odyssey Infrared Imaging System (LI-COR; ScienceTec, Les Ulis, France). The expression of phosphorylated p38 MAPK was estimated after normalization with β-actin.
The expression of phosphorylated p38 MAPK in IPEC J2 cell lysates was analyzed by western blot as already described26 (link). Briefly, total proteins were separated on SDS-PAGE, transferred onto nitrocellulose membranes, incubated with Rabbit anti-phospho-p38 (Cell Signaling Technology, Danvers, MA) overnight and further incubated with CFTM770 goat anti-rabbit IgG (Biotium, Hayward, CA). Mouse anti-β-actin (Cell Signaling Technology) was used as control. Membranes were analyzed using an Odyssey Infrared Imaging System (LI-COR; ScienceTec, Les Ulis, France). The expression of phosphorylated p38 MAPK was estimated after normalization with β-actin.
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