Fig. 3: Cathepsins are dispensable for NF-kB-mediated responses related to the NAIP/NLRC4 priming step.

A Starch-elicited peritoneal macrophages (PMs) isolated from C57BL/6 WT mice were treated with the cathepsin B inhibitor Ca-074Me (25 μM) for 1 h. Then, PMs were primed with LPS (200 ng/ml, 3 h) and incubated with empty DOTAP or with ultrapure flagellin extracted from Salmonella typhimurium inserted into DOTAP (FliDot) (1 μg/ml). The release of the active form of IL-1β was detected by western blot of the culture supernatant (Sup). Pro-IL-1β and β-actin were detected by western blot of the cellular lysates (Lys). Densitometry analysis was performed using ImageJ software and the relative expression of IL-1β was normalized by pro-IL-1β. Data representative of two-independent experiments. B Bone marrow-derived macrophages from C57BL/6j WT mice were pretreated with the cathepsin B inhibitor Ca-074Me, primed with LPS and stimulated with FliDot. TNF-α production was evaluated in the culture supernatants by HTRF. The bars represent the average of three independent experiments performed in technical triplicates ± SD. C PMs were pretreated with the cathepsin B inhibitor Ca-074Me for 1 h and stimulated with FliDot for the indicated periods. The phosphorylation of NF-κB were analysed by Western Blotting. β-actin served as reference protein. Data representative of three-independent experiments.