Alphascreen surefire erk1 2 assay kit

Cells were transferred to serum-free medium 2 h prior to assay. Ligands and controls (10 µl of 10× concentrates) were added to 90 µl medium and incubated for 5 min at 37 °C. The final concentration of DMSO was 0.2%. The reaction was stopped by aspiration of the medium and addition of 50 µl lysis buffer (PerkinElmer Life Sciences). To measure ERK1/2 phosphorylation, the AlphaScreen^®SureFire ERK 1/2 assay kit (PerkinElmer Life Sciences) was used with the high sensitivity protocol. Phosphorylated ERK1/2 was measured in 384-well white OptiPlate microplates (PerkinElmer Life Sciences) with the Fusion AlphaScreen multilabel reader (PerkinElmer Life Sciences).

KNRK and KNRK-PAR2 cells were seeded into 96 well plates (3×10⁵ cells/well). After 24 h, cells were serum starved in 80 μl phenol red free DMEM for at least 12 h to reduce basal ERK activity. Cells were challenged with 10 μl of buffer, GzmK (100 nM), with or without 30 min pre-treatment of EGFR inhibitor AG1478 (100 nM), inactivated GzmK (100 nM), trypsin (10 nM), FBS (10%) for 5 min. Concentration response measurements were also conducted for trypsin and GzmK at increasing protease concentrations. ERK1/2 phosphorylation was detected using the AlphaScreen SureFire ERK1/2 assay kit (PerkinElmer) according to the manufacturer’s instructions.

ERK1/2 phosphorylation was detected with the AlphaScreen SureFire ERK1/2 assay kit (PerkinElmer). Briefly, GHRHR or SV1 expressing HEK293T cells were seeded into 96-well culture plates coated with poly-D-lysine (30,000 cells/well) and grown overnight followed by deprivation of serum for at least 6 h. After incubation with FBS-free DMEM medium containing 4 μM GHRHR antagonist (MIA-602) or vehicle control for 30 min at RT, ERK1/2 phosphorylation was stimulated by the addition of 1 μM GHRH (100 μL final volume) at the indicated time points. GHRH stimulation was terminated by removal of medium and addition of 30 μL of SureFire lysis buffer to each well. The plate was then agitated for 15 min. A 1:17:100 (vol/vol/vol) dilution of AlphaScreen beads/SureFire activation buffer/SureFire reaction buffer was transferred to a white 384-well Proxiplate (8.5 μL per well) followed by addition of 5 μL lysate in diminished light. The plate was incubated in the dark at 37 °C for 1 h, after which the fluorescence signal was measured by an Envision plate reader using standard AlphaScreen settings. Data were normalized to the maximal response elicited by 10% FBS for 7 min followed by normalization to the maximal response elicited by GHRH.