Fast-Activated Cell-Based Signaling Assay

Fast-activated cell-based (FACE) assays were performed as we described previously [92 (link)]. SFs were seeded at 10⁴ cells/well (96-well plates) and after overnight incubation in 1% FCS DMEM to synchronize the cells, were then stimulated for the indicated time points (from 0 to 120 minutes) before being treated with fixative buffer (Biolegend) according to the manufacturer’s recommendations. Once permeabilized, the cells were incubated with antibodies specific for either the activated or whole protein form of the signalling element (e.g., ERK1/2 and the dually phosphorylated active forms of ERK [pERK; Cell Signalling Technology]) for 1 hour before addition of streptavidin-conjugated horseradish peroxidase for 30 minutes (anti-rabbit HRP, Cell Signalling Technology). Following washing and incubation with tetramethylbenzidine (TMB), absorbance was read at 450nm using a Tecan Sunrise plate reader.