Ectopic expression of COP1 in 32D cells induces degradation of C/EBPα and blocks granulocytic differentiation in response to G-CSF. (A) 32D cells were infected with viruses containing GFP and GFP-COP1 and selected in the presence of puromycin. Lysates from GFP-positive cells were analyzed by western blotting using antibodies against COP1, C/EBPα, and γ-tubulin. (B) 32D transfectants (GFP and COP1) were incubated in the presence and absence of MG132 and analyzed by immunoblotting with antibodies to C/EBPα and γ-tubulin. The relative amounts of C/EBPα are presented as the ratio of C/EBPα and γ-tubulin, and calculated with the level of untreated 32D cells (GFP control) as 1.0. (C) 32D transfectants (GFP and COP1) prepared in A were cultured in IL3, transferred to G-CSF for 4 days, and analyzed for expression of Mac-1 by flow cytometer. To confirm the expression of GFP, results with the dot blot analysis are also shown. Results shown in A-C are representative of 2 independent experiments. (D) 32D transfectants (GFP and COP1) were cultured in IL3 and transferred to G-CSF for 4 days. BrdU incorporation and DNA content were analyzed by flow cytometer. Quantification of the data, average of 2 independent experiments, is also shown as means ± standard deviation (SD) at the bottom.