Fig. 4

Development and validation of a MEK5A/MAPK7 cell-based in vitro pharmacodynamic ELISA co-expression assay. a Western blot analysis of HEK293 cell transfectants. HEK293 cells were transfected with plasmid vectors and/or treated with compound as indicated in the matrix (details in ‘Methods’ section). Cell lysates were prepared and analysed using Western blot to detect phospho-MAPK7 (T218/Y220), total MAPK7, HA-tag and GAPDH. b Using the same cellular lysates, a quantitative sandwich ELISA assay was developed by coating ELISA plates with a total MAPK7 capture antibody, incubating with lysate and then using a phospho-MAPK7 (T218/Y220) detection antibody. Phospho-MAPK7 signals were then quantified using an envision reader. c Further validation of the pMAPK7 ELISA assay was performed by running dose–response experiments with 4 small molecules; 2 MAPK7 inhibitors and 2 unrelated kinase inhibitors (AZD2281 and AZD3965)