Figure 5.

MEK-ERK activation is necessary but not sufficient for induction of cathelicidin expression in both skin and gut epithelial cells. (a) Keratinocytes (HaCat) were stimulated with 1,25(OH)₂ VD₃ with or without prior incubation with the MEK-ERK inhibitor U0126 (20 μm, 30 min) and cathelicidin expression assessed by real-time RT-PCR. (b) HT-29 colon cells were stimulated with butyrate with or without prior U0126 treatment and cathelicidin expression was analysed. Data shown are means (± SD) of a single experiment performed in triplicates and are representative of three independent experiments. (**P < 0·01; Student's t-test). (c) To determine if MEK-ERK activation was critical for cathelicidin induction keratinocytes were stimulated with the control (lane 1), 1,25(OH)₂ VD₃ (lane 3) or EGF (lane 5) for 15 min with or without prior incubation with U0126 (lanes 2,4,6) and subjected to SDS–PAGE and Western blot. (d) HT-29 colon cells were stimulated with the control (lane 1), butyrate (lane 3) or EGF (lane 5) for 15 min with or without prior incubation with U0126 (lanes 2,4,6) and analysed by Western blot. As a positive control a phosphorylated ERK protein was used on both blots (lane 8). Blots were stained with a specific phospho-ERK antibody and visualized by chemiluminescence.