Cell-based ELISA for phosphorylated ERK

Phosphorylated ERK was measured according to a protocol described previously (Versteeg et al., 2000). Stable HEK293 cell lines were seeded at density of 3 × 10⁵ per well in 96-well plates and grown overnight. Cells were treated for 30 min followed by removal of media and fixation in 4% formaldehyde in PBS for 20 min at room temperature. Fixed cells were permeabilized by three washes in 0.1% Triton X-100 in PBS (PBST), and endogenous peroxidase activity was quenched by 20-min incubation in PBS containing 0.6% H₂O₂. Following three washes in PBST, samples were blocked (10% FBS in PBST) for 1 h and incubated overnight with primary antibodies against phospho-ERK1/2 (Cell Signaling Technology #9101) at 1:500. Samples were washed three times in PBST and twice in PBS. An HRP-coupled anti-rabbit secondary antibody (CST #7074), diluted 1:500 in PBST containing 5% BSA, was added for 1 h at room temperature. Following five washes in PBST, samples were exposed to the colorimetric substrate One-Step Ultra TMB-ELISA (Pierce, catalog 34028). After development in the dark for 10 min, the reaction was stopped by adding 4 M sulfuric acid, and absorbance was read at 450 nm.