Fig. 4

Signalling downstream of S100A8 and S100A9. A] Panc-1 cells were treated with [2 μg/mL] of recombinant A8-GST, A9-GST, A8-GST + αA8, A9-GST + αA9, A8-GST + αRAGE and A9-GST + αRAGE for 60 min and probed by Western blotting for the indicated phosphoproteins. β-actin served as a loading control. B] Panc-1 cells were transfected with NF-κB and control reporters [2 μg of DNA] and stimulated with recombinant A8-GST, A9-GST or GST [2 μg/mL] for 24 h. Cells treated with TNF-α [10 ng/mL] served as a positive control for NF-κB induction. For blocking conditions, cells were treated with anti-RAGE blocking antibody for 1 h prior to stimulation. S100A8-GST and S100A9-GST were incubated with anti-S100A8 and anti-S100A9 neutralising antibodies for 1 h before addition to cancer cell cultures. Luciferase activity was plotted as the mean ± SEM of three independent experiments, each performed in triplicate. Error bars represent standard error (* P < 0.05)