The Trib1 regions required for enhanced self-renewal activity of bone marrow cells. (A) Constructs of Trib1 mutants. NLS indicates a nuclear localization signal. (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) Replating assay. Primary murine bone marrow cells were infected with retroviruses bearing the indicated mutants. The colony numbers in methylcellulose culture during 4 serial replatings were measured. The means ± SDs from 3 independent experiments are shown (left). Representative results of methylcellulose cultures are shown at center. Expression of FLAG-tagged Trib1 mutants in packaging cells was detected by immunoblotting with anti-FLAG mAb (right).