Journal:

Article Title: Regulation of induction of nitric oxide synthase and the inhibitory actions of dexamethasone in the human intestinal epithelial cell line, Caco-2: influence of cell differentiation

doi: 10.1038/sj.bjp.0702827

Figure Lengend Snippet: Inducible NOS protein expression in Caco-2 cells assessed by Western blot. The inducible NOS protein expression was determined by Western blot using a polyclonal iNOS antibody. Each panel is a representative of three different experiments. (a) Caco-2 cells (passages 32 to 39) were used 10 days after confluence. Cells were treated for 18 h with vehicle alone (Con), cytomix (CM), cytomix and cycloheximide (CHX) (5 μg ml−1, added concurrently), cytomix and actinomycin D (ActD) (5 μg ml−1, added concurrently), or cytomix and pyrrolidine dithiocarbamate (PDTC) (200 μM, 2 h pre-treatment). (b) Caco-2 cells were used 10 days after confluence. Cells were treated for 18 h with vehicle alone (Con), cytomix (CM) or cytomix and dichloroisocoumarin (DCI) (200 μM, 2 h pre-treatment). (c) Caco-2 cells were used 10 or 15 days after confluence as indicated. Cells were treated for 18 h with vehicle alone (Con), cytomix (CM), or cytomix and dexamethasone (Dex) (5 μM). (d) Caco-2 cells were used 10 days after confluence. Cells were treated for 18 h with vehicle alone (Con), cytomix (CM), or cytomix and tyrphostin A25 (200 μM), cytomix and tyrphostin B42 (200 μM), cytomix and PDTC (100 μM, 2 h pre-treatment), or cytomix, PDTC (100 μM) and A25 (20 μM).

Article Snippet: After blocking with PBS (pH 7.4), 0.25% tween 20 (v v −1 ) and 5% non-fat dried milk, membrane was probed with anti-iNOS polyclonal antibody (1/500) (Autogen Bioclear, Calne, U.K.) for 1 h at room temperature, washed with PBS-tween 20 and then incubated with horseradish peroxidase-conjugated second antibody (1/4000) for 1 h at room temperature.

Techniques: Expressing, Western Blot