MLF1 inhibits the polyubiquitination of C/EBPα by interfering with Trib1-COP1 complex formation. (A) 293T cells were transfected with a combination of vectors shown at the top. Cell lysates were analyzed by immunoblotting with antibodies to a FLAG epitope, MLF1, COP1, an HA epitope, and γ-tubulin. (B) 293T cells were transfected with a combination of vectors shown at the top. Cell lysates were analyzed by immunoprecipitation with an antibody to a FLAG epitope followed by immunoblotting with an antibody to polyubiquitin chains (poly-Ub), and by immunoblotting with antibodies to COP1, MLF1, and a FLAG epitope. Total RNA was analyzed by semi-qRT-PCR using a pair of primers specific to Trib1 and human GAPDH. (C) 293T cells were transfected with a combination of vectors shown at the top. Cell lysates were analyzed by immunoprecipitation with an antibody to an HA epitope followed by immunoblotting with antibodies to an HA epitope, MLF1, and COP1.