A floxed neo cassette was inserted within intron 4, and specific mutations in exon 3 were introduced by homologous recombination. Mutations resuted in three sites of MAPK phosphorylation (Ser-55, Ser-65, and Ser-73) in exon 3 to be substituted with alanines (S55A, S65A, S73A). The neo cassette was then excised with cre recombinase leaving a single loxP site in intron 4. Expression of normal amounts of mutant proteins was confirmed by immunoblot.