Inhibiting EGFR and ERK1/2 Signaling Pathways

Chemical inhibitors of MEK, U0126 and PD98059 (Cell Signaling Technology), were used at concentrations of 10 and 50 μM, respectively. Bay43-9006 (Cayman Chemical), a chemical inhibitor of Raf, was used at 15 μM. Retro2 (Sigma–Aldrich), a retrograde trafficking inhibitor, was used at a concentration of 100 μM (Nelson et al., 2013 (link)). All chemical inhibitors were reconstituted in DMSO (Cell Signaling Technology), which served as a volume-specific vehicle control. EGFR and ERK1/2 siRNAs (Cell Signaling Technology) were transfected into SVG-A cells with RNAiMax (Thermo Fisher) at 10 pmol per well per manufacturer’s instructions. Successful transfections of the siRNAs were confirmed using BLOCK-iT Red (Thermo Fisher) (DuShane et al., 2018 (link)).