Degradation of C/EBPα by Trib1 is dependent on the MAP kinase pathway. (A) Interaction between Trib1 and C/EBPα. 293 cells were transiently transfected with FLAG-tagged Trib1 and HA-tagged C/EBPα. The expression level of each protein was assessed by immunoblotting whole cell lysates with anti-HA or anti-FLAG antibodies. (B) NH112 cells were infected with the indicated FLAG-Trib1 retroviruses. The cell lysates were immunoblotted with indicated antibodies. (C) NH112 cells infected with an empty retrovirus (vector) or the Trib1 retrovirus (Trib1 wt) were treated with cycloheximide with/without U0126 for the indicated periods. The cell lysates were immunoblotted with the indicated antibodies. The relative intensities of C/EBPα bands compared with β-tubulin bands are measured (bottom). The values are means ± SE s from 4 independent experiments. Statistical signicicances were confirmed using the Mann-Whitney test with the Dunn-Sidak post hoc test (*P = .026, **P = .00866), and 1-way analysis of variance for repeated measures with the Dunnett test was also performed. Full description for the statistical analysis is available as supplemental information. (D) Phosphorylation status of C/EBPα serine 21 was assessed in NH112 cells infected with an empty retrovirus (vec), wild-type Trib1 (wt), ΔN2 mutant (ΔN2), or ΔILLHPWF (ΔILL) mutant retroviruses. The expression of total C/EBPα is shown at the bottom with β-tubulin as a loading control, and phosphorylated C/EBPα (pC/EBPα) in equal amounts of total C/EBPα is exhibited at the top panel.