Figure 7: Upstream and downstream components of the CARD9 signalling pathway.
(a–c) Wild type (WT), Hck−/−Fgr−/−Lyn−/− (3 × SFK KO), Syk−/−, Plcg2−/− or Card9−/− neutrophils were placed on immobilized immune complex (IC) surfaces. Their superoxide release (a), MIP-2 levels (b) and LTB4 (c) release were determined. (d) WT and Card9−/− neutrophils were plated on IC surfaces for 10 min, the phosphorylation and the total amount of Syk in cell lysates was determined by western blot after immunoprecipitation. (e–h) Bcl10−/− and Malt1−/− neutrophils showed similar functional phenotypes on IC surfaces like Card9−/− neutrophils: they had intact superoxide release (e,g) and defective chemokine production (f,h). (i) WT and Card9−/− neutrophils were stimulated for 20 min; the phosphorylation and degradation of IκBα was followed by western blotting from whole cell lysates. (j) IC-activated WT and Card9−/− neutrophils were lysed after 20 min; the activation and nuclear translocation of NF-κB was determined by infrared dye-labelled NFκB consensus binding site probes. Kinetic curves in a,e and g show mean and s.e.m. of three to four independent experiments. Control data points were subtracted. Graphs in b,c,f and h represent data from two to four independent experiments. (d,i,j) Representative of two to three independent experiments. *P<0.05; NS, statistically not significant (two-way ANOVA); see the text for actual P values.
