FPRL1- and MARCO-mediated ERK1/2 phosphorylation and changes of cAMP levels in transfected HEK293 cells. For analysis of ERK1/2 phosphorylation, untransfected (A) or hMARCO- (B), hFPRL1- (C), and hFPRL1/hMARCO- (D) expressing HEK293 cells were treated with Neisseria meningitidis (NM), Streptococcus pneumoniae (SP) or 1 μM fMLF for 5 and 15 min at 37°C. Cells were lysed, equal amounts of protein (5 μg) were dissolved by SDS sample buffer, and levels of total ERK2 and phosphorylated ERK1/2 were determined via immunoblotting. The positions of molecular mass markers are indicated on the left (in kDa). The mean ± SD of the three independent experiments were evaluated by densitometric quantification normalized to ERK2 expression (E to H). Asterisks indicate a significant difference (*, p < 0.05; **, p < 0,01; ***, p < 0,001) compared to control (one-way ANOVA followed by the Bonferroni test). For analysis of inhibition of forskolin-stimulated adenylate cyclase activity, untransfected (I) or hMARCO- (J), hFPRL1- (K), and hFPRL1/hMARCO- (L) expressing HEK293 cells were subjected to 25 μM forskolin as well as to NM or 1 μM fMLF for 15 min at 37°C. cAMP levels were determined as described above (see Methods). The values represent mean ± SEM from four independent experiments. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0,01) between forskolin plus agonists and forskolin alone, as determined via one-way ANOVA followed by the Bonferroni test.