Fig. 3.

RAF kinases are dimerized in moDCs in response to LPS. a Immature moDCs were stimulated for 1 and 24 h with LPS (100 ng/ml) and phosphorylation of CRAF (S338), MEK (S217/S221), and ERK1/2 (Y202/Y204) was determined by western blot analysis. b The relative level of CRAF (n = 3), c MEK1/2 (n = 4), and d ERK1/2 phosphorylation (n = 3) was determined by the ratio of phosphorylated protein to total protein levels. e Endogenous RAF proteins were immunoprecipitated from immature moDCs and from LPS-stimulated moDCs (100 ng/ml, 1 h and 24 h). Immunoprecipitates were analyzed by western blot. The data are representative for four independent experiments. Arrows are highlighting the observed RAF-interactions. f Kinase activity of RAF proteins immunoprecipitated from immature moDCs and LPS-stimulated moDCs (100 ng/ml, 1 h and 24 h) was studied as mentioned in the “methods” section. Phosphorylation of the kinase dead MEK K97A substrate at S217/S221 was investigated by western blot and a representative experiment is shown