HEK293 cells were transiently transfected with HA-tagged wild-type (WT) MEK1, its single-nucleotide polymorphism (SNP) variant (E44G) that is found in healthy individuals18, its clinically-identified mutants found in RASopathies (hereafter referred to as Rmuts) (F53S, T55P, D67N, and Y130C)18 or in sporadic cancers (hereafter referred to as Cmuts) (Q56P, K57N, C121S, and E203K)13, or a constitutively active, phosphomimetic MEK1-S218D/S222D (DD) mutant, together with kinase-dead Myc-ERK2(K/N) as a substrate. The gene discussed is MAP2K1; the disease is RASopathy.