Identification of the Trib1 motif required for MEK1 binding, enhanced ERK phosphorylation and self-renewal activity. (A) Constructs of Trib1 mutants and a conserved amino acid motif in Trib family proteins. (B) HeLa cells were transiently transfected with FLAG-tagged Trib1 and HA-tagged MEK1. The cell lysates were immunoblotted with an anti-HA polyclonal antibody. The expression level of each protein was assessed by immunoblotting whole cell lysates with anti-HA or anti-FLAG antibodies. (C) Mouse primary bone marrow cells were infected with indicated FLAG-Trib1 retroviruses. The cell lysates were immunoblotted with the indicated antibodies. (D) Replating assay. Primary bone marrow cells were infected with the indicated retroviruses. The colony numbers in methylcellulose culture during 3 replatings were measured. The means ± SDs from 3 independent experiments are shown (left). The representative results of methylcellulose culture are shown (right).