RAF kinase assay

Endogenous RAF proteins were immunoprecipitated from human immature moDCs, from moDCs stimulated with LPS (1 and 24 h) and from cells differentiated to moDCs for 2–5 days. Alternatively, RAF proteins were immunoprecipitated from moDCs treated for 6 h with DMSO or LY3009120. In both cases, IgG control was included. Further, kinase activity of V5-tagged ARAF WT and ARAF mutants (ARAF Y301D/Y302D, ARAF R362H, ARAF S257A, and ARAF S257D) was investigated. Constructs were overexpressed in HEK293T cells and immunoprecipitated via the V5 tag. The empty vector served as control. Agarose-coupled protein A/G beads were employed to precipitate the antibody/antigen complexes to which a reaction mix of 1× kinase buffer [10× buffer: 100 mM MgCl₂, 250 mM β-glycerolphosphate, 250 mM HEPES pH 7.5, 50 mM benzamidine, 5 mM DTT, 10 mM NaVO₃; diluted to 1× with H₂O] and 1 µg of kinase dead His-MEK1 K97A (Cat. No. M02-16H, SignalChem) was added in a total volume of 38 µl. After adding MgATP (Enzo Life Sciences; stock: 20×; diluted to 1×), the reaction was incubated at 30 °C for 30 min. Kinase assays were stopped by adding 10 µl of Laemmli buffer and loaded onto an SDS-PAGE gel for immunoblot analysis.