p38 and JNK mediate ApxI-induced proinflammatory cytokine production. (A) PAMs were stimulated with 0.5 CU/mL of ApxI, heat-inactivated ApxI (Δ), or control medium (C) for 1 h. Subsequently, cells were lysed and subjected to Western blot analysis using an antibody specific to phospho-p38 (p-p38) or phospho-JNK (p-JNK). (B-D) PAMs were pre-incubated with a p38 inhibitor SB203580 (SB), a JNK inhibitor SP600125 (SP), or vehicle DMSO (V) for 1 h, followed by stimulation with 0.5 CU/mL ApxI for 2 h. PAMs were subjected to RT-qPCR analysis for mRNA levels of IL-1β (B), IL-8 (C), and TNF-α (D). (E) PAMs were pre-incubated with SB, SP, or DMSO for 1 h, followed by stimulation with 0.5 CU/mL of ApxI for 8 h. Subsequently, the culture supernatants were collected and the protein levels of IL-1β, IL-8, and TNF-α were determined by ELISA. Data are representative of three independent experiments of at least triplicate determinations. ***p < 0.001.
Chen et al. Veterinary Research 2011 42:25 doi:10.1186/1297-9716-42-25