AlphaScreen Analysis

The inhibition of ERK phosphorylation was examined with the bead-based amplified luminescent proximity homogeneous assay (AlphaScreen). The assay was used, according to manufacturer’s instructions, to detect phosphorylated ERK (SureFire p-ERK 1/2, (Thr202/Tyr204), PerkinElmer, Rodgau, Germany). MCF-7 and MDA-MB-468 cells were seeded at 2 × 10⁴/well in 96-well plates. Cells were pre-treated with ChPL and/or AG1478 (1 μM) for 1 h followed by a stimulation with EGF (epidermal growth factor) (50 ng/ml) for 30 min in 5% CO₂ at 37°C. Cells were lysed by the addition of 50 µl of SureFire lysis buffer (PerkinElmer) and plates were incubated at room temperature with agitation for 10 min (∼350 rpm). To determine p-ERK1/2 levels, cell lysates (4 µL) were transferred to a 384-well plate (Proxiplate, PerkinElmer) followed by the addition of the Reaction mix (7 µl) containing the Reaction buffer, Activation buffer, and Acceptor and Donor beads. Plates were incubated for 2 h at room temperature. The fluorescent signals were read with an Envision Multilabel Reader (PerkinElmer) with standard AlphaScreen settings.