Trib1 cooperates with COP1 to induce ubiquitination of C/EBPα and block granulocytic differentiation. (A) Total RNA was extracted from cells indicated on the top and analyzed by quantitative RT-PCR using a pair of primers specific to Trib1 and Trib2. (−), negative control. (B) 32D cells were coinfected with control and untagged COP1-containing viruses (the pMSCV-IRES-GFP retroviral vector [MigR1]) together with viruses containing control siRNA (siGL2) and siRNA specific to Trib1 (siTrib1). Cell lysates were subjected to immunoblotting with antibodies to COP1 and γ-tubulin. Total RNA was analyzed by quantitative RT-PCR using a pair of primers specific to Trib1. Quantitative data are shown. (C) 32D transfectants prepared as in B were maintained in IL3, transferred to G-CSF for 4 days, and analyzed for expression of Mac-1 by flow cytometer. (D) BM cells were fractionated into different lineages shown at the top, lineage marker (Gr-1, Mac-1, TER119, B220, and CD3ε) positive hematopoietic cells (Lin⁺), CD34⁻Lin⁻Sca-1⁺c-Kit⁺ (LSK) hematopoietic stem cells (HSCs), CD34⁺LSK multipotent progenitors (MPPs), FcγRII/III^loCD34⁺ common myeloid progenitors (CMPs), FcγRII/III^hiCD34⁺ GMPs, and FcγRII/III^loCD34⁻ megakaryocyte-erythroid progenitors. Control, positive control for C/EBPα and COP1. −, negative control. Total RNA extracted from each cell was analyzed by quantitative RT-PCR using a pair of primers specific to C/EBPα, COP1, Trib1, Trib2, and β-actin. (E) BM cells were fractionated into different lineages by the surface markers shown at the top, Lin*^negSca-1⁻Mac1^lo/+c-Kit⁺ cells (Lin*, lineage marker without Mac-1 and Gr-1) containing committed myeloid progenitors, Lin*^negSca-1⁻Mac1^+/hic-Kit^lo cells (differentiated immature myeloid cells), and differentiated population of cells positive for each lineage marker (granulocytes; Gr-1, macrophages; Mac-1, erythrocytes; TER119, B lymphocytes; B220, and T lymphocytes; CD3ε). Total RNA extracted from each cell was analyzed by quantitative RT-PCR using a pair of primers specific to COP1, Trib1, Trib2, and β-actin. Control, positive control for COP1, Trib1, and Trib2. (−), negative control. (F) GST and GST-COP1 proteins produced in Escherichia coli were immobilized on glutathione beads and incubated with crude cell extracts containing HA-tagged Trib1 protein. The bound protein was detected by immunoblotting with antibody to an HA-epitope. GST-fused proteins were visualized by Coomassie brilliant blue staining. (G) NIH3T3 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 a HA epitope, by immunoblotting with an antibody to COP1, and by quantitative RT-PCR using a pair of primers specific to Trib1 and β-actin. Results are representative of 3 independent experiments. Vertical lines have been inserted to indicate a repositioned gel lane (D-E and G).