Fig. 2

Effect of sialostatins on the signalling pathways activated by LTA in dendritic cells. Dendritic cells were seeded in 24-well plate. Next day DCs were incubated 2 h with tick cystatins (both 3 μM) prior to the addition of LTA (2 μg/ml) and further incubated for indicated times. Afterwards, cells were lysed and obtained protein extract was further analysed by immunoblotting using antibodies recognizing phosphorylated form of tested kinases. Membranes were stripped and reprobed with antibodies against total kinase protein, which served as a control (a). Proteins were visualized by enhanced chemiluminescence. Bands were quantified using scanning densitometry and phosphorylation/activities of Erk1/2 (b), Akt (c), and NF-κB (d) kinases were normalized by total kinase protein level (relative activity = phospho kinase/total kinase). Three independent experiments were performed and representative blots are shown. Graphs represent the average ± SD from 2-3 experiments, the relative phosphorylation of kinase achieved at 60 min upon LTA stimulation was set up to 1 to allow pooling of data