Figure 3.

C3d activation of the PI3K/Akt–NF-κβ pathway. (A) Expression levels of AKT post C3d (10 µg/1×10⁵ cells) treatment was measured by western blot analysis of monocyte whole cell lysates. Controls included preincubation with BAY 11-7082 (NF-κβ inhibitor, 5 µg/mL), CR3 blocker (clone mAb 107, 1 µg/mL), LY-294002 (LY) PI3 kinase inhibitor, 10 µM) or isobavachalcone (Isob) (AKT inhibitor, 5 mM) for 30 min prior to C3d challenge. Phosphorylation levels were normalised to respective total protein. Results are expressed as DU, with representative western blots presented (n=6 biological repeats, one-way ANOVA, followed by Tukey’s posthoc multiple comparison). (B) The effect of C3d (10 µg/mL) on pro-IL-1β mRNA levels by qPCR analysis and protein levels by ELISA (C) with or without the inclusion of CR3 and NF-κβ blockers (n=6 biological replicates, one-way ANOVA, followed by Tukey’s posthoc multiple comparison test). Data are expressed as mean±SD. DU, densitometry units; IL-1β, interleukin-1β; LPS, lipopolysaccharide.