ERK1/2 Phosphorylation Assay (p-ERK)

PBMC were pelleted by centrifugation for 10 min at 250g, resuspended in assay medium, and seeded at 1 × 10⁵ cells in 10 μL per well into Falcon 384-well plates. Plates were incubated in a humidified atmosphere at 37 °C and 5% CO₂ for 55 min, and then transferred to the surface of a water bath at 37 °C and incubated for 5 min prior to adding ligands or vehicle controls. Treatments were prepared at 2× concentrations in assay medium, and dispensed at 10 μL per well at requisite time points. At the end of incubation, plates were placed on ice, and cells were lysed by the addition of 10 μL per well of 3× concentrated ice-cold lysis buffer (formulation as per AlphaLISA kit described below). The plates were placed on shakers (500 rpm, 10 min at 4 °C) and frozen at −80 °C. p-ERK detection was performed using the AlphaLISA SureFire Ultra p-ERK 1/2 (Thr202/Tyr204) Assay Kit (PerkinElmer) per the manufacturer’s specifications in ¹/₂-area white 96-well plates. The signal was detected on a CLARIOstar plate reader with AlphaLISA-compatible filters. Counts were normalized to vehicle controls (100%) to allow compilation of data from independent experiments.