MEK1 VUS validation experiments

MEK1 mutant constructs were generated from the C-terminal GFP-tagged MEK1 plasmid (pEGPF-N1-MEK1, Addgene, #14746) using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) and verified by Sanger sequencing. HEK-293H cells were transiently transfected with the wild-type or mutant MEK1-GFP plasmid using Lipofectamine® 2000 Transfection Reagent (Invitrogen). At 24 hr post-transfection, media was aspirated and refreshed for 1 hr at which time cells were harvested, lysed in 1% NP-40 buffer with protease and phosphatase inhibitors, protein quantified, and processed for immunoblotting. Rabbit polyclonal antibodies recognizing phosphorylated MEK1/2 (Ser217/221), MEK1/2, phosphorylated ERK1/2 (Thr202/Tyr204), and ERK1/2 were obtained from Cell Signaling. Rabbit monoclonal antibodies recognizing GFP and GAPDH were obtained from Cell Signaling. After incubation with horseradish peroxidase-conjugated secondary antibody, proteins were detected by chemiluminescence (SuperSignal West Pico Plus Chemiluminescent Substrate, Thermo Scientific) and visualized using the Fuji LAS-4000 imager (GE Life Sciences).