Figure 1

ILK is induced by pro-inflammatory stimuli. A. Colonic SW480 cells were stimulated with 1 ug/ml LPS for 24 h, and after harvesting the cells, western immunoblotting was performed. Membranes were probed with antibodies for ILK, ser473Akt, Akt and beta-actin. ILK protein induction is accompanied by an increase in the phosphorylation of Akt at ser473, but not the protein level of this kinase. The beta-actin is shown as an internal control. B. This shows the response of a murine colonic explant to exposure with 2.5% DSS for 48 h. There is an increase in the expression of ILK. The lower panel shows equivalent levels of beta-actin. C. Colons of control and DSS treated mice (48 h) were probed with a polyclonal antibody to ILK. Besides an increase in the intensity of the signal generally, more than 50% of the epithelial cell nuclei stain positively for ILK. D and E. Colonic HCT116 cells were stimulated with 5 ng/ml IL-1β for 4 h. Protein was obtained by lysing cells in homogenization buffer and western blotting performed for ILK expression (D). Alternatively, RNA was obtained by Trizol and used for cDNA synthesis; semi-quantitative RT-PCR for ILK was then performed using the primers indicated in materials and methods. Wortmannin and Ly294002 were both capable of inhibiting the IL-1β-induced ILK protein and mRNA. The β-actin signal is shown as the internal control. F. The experiment was repeated as in D with the addition of inhibitors of the MAPK pathway. Specifically PD98059, SP600125 and SB203580 were used to inhibit the p42/44 ERK, P54/45 JNK and p38SAPKs respectively, and GAPDH used as the loading control. Data are representative of 3 independent experiments for each panel.