Immunoblotting of MAPK Pathway Proteins

Immunoblotting was performed as previously described (Brian et al., 2022 (link); Brian et al., 2020 (link)). Briefly, 0.025 million cell equivalents of whole cell lysate were run through 7% Tris-acetate polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked in Intercept (TBS) Blocking Buffer (LI-COR Biosciences) for 1 hr, and then incubated overnight with primary antibody: ARAF (Cell Signaling), BRAF (Cell Signaling), CRAF (Cell Signaling), MEK1/2 (Cell Signaling), and ERK1/2 (Cell Signaling). Membranes were washed and incubated for 1 hr at room temperature with corresponding species-reactive secondary antibody and then imaged using an Odyssey CLx near-infrared imager (LI-COR Biosciences).