![Figure 3. Different ability of splicing variants of COP1 to induce degradation of C/EBPα and block granulocytic differentiation. (A) NIH3T3 cells were transfected with vectors containing GFP-fused full-length COP1 (WT) and splicing variants (Δ4, Δ20, and Δ24) shown in the schematic diagram (top). Cells were viewed under a fluorescence microscope. Cells transfected with untagged COP1 were stained with antibody to COP1 and visualized with an FITC-labeled secondary antibody. PC, phase contrast. (B) Lysates from GFP-positive 32D cells infected with viruses containing GFP, GFP-COP1 (full-length [WT] and splicing variants [Δ4, Δ20, and Δ24]) were analyzed by western blotting using antibodies against COP1, C/EBPα, and γ-tubulin. (C) 32D transfectants prepared in B were maintained in IL3, transferred to G-CSF for 4 days, and analyzed for expression of Mac-1 by flow cytometer. Results are representative of 2 independent experiments. (D) RT-PCR products generated by use of template RNAs isolated from 32D cells and control NIH3T3 cells were digested with BstXI or DraIII. A lower panel shows the sizes of the digested fragments of WT COP1 and alternatively spliced variants.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/122/10/10.1182_blood-2012-12-476101/4/1750f3.jpeg?Expires=1735831494&Signature=fdbH8xFo8awy7xlaFkepG60ZFJc9uq1vvH6WiXTmTdNI8BIYwj2ysYOvV~rdCfRpNQBj4~CqXtMmpliXO-91c6nJAs961eLEFmrGxtNXOfltGGvK~Qj2qm4M-qb4kQeTYnaQlsSkrqfLw9hvJfyNRnIsIzSOWcNPIBZCqNv4hxIvLGbq8HeH9jI1W45wT44vNBdB2U7OZPh6JKOxOSEhoEcmr5HDdxZ322~4DcuNR~g9~vEIV1TUOxLQzX3c~J97qfJCmoNYP4Du8gh-9v7K6Osbp9W7g5Kd-gOJVONNJfgCMLMsukdKNGmfj99a3cMHGs-uRS-vx9S9dmS1qc6Jow__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Different ability of splicing variants of COP1 to induce degradation of C/EBPα and block granulocytic differentiation. (A) NIH3T3 cells were transfected with vectors containing GFP-fused full-length COP1 (WT) and splicing variants (Δ4, Δ20, and Δ24) shown in the schematic diagram (top). Cells were viewed under a fluorescence microscope. Cells transfected with untagged COP1 were stained with antibody to COP1 and visualized with an FITC-labeled secondary antibody. PC, phase contrast. (B) Lysates from GFP-positive 32D cells infected with viruses containing GFP, GFP-COP1 (full-length [WT] and splicing variants [Δ4, Δ20, and Δ24]) were analyzed by western blotting using antibodies against COP1, C/EBPα, and γ-tubulin. (C) 32D transfectants prepared in B were maintained in IL3, transferred to G-CSF for 4 days, and analyzed for expression of Mac-1 by flow cytometer. Results are representative of 2 independent experiments. (D) RT-PCR products generated by use of template RNAs isolated from 32D cells and control NIH3T3 cells were digested with BstXI or DraIII. A lower panel shows the sizes of the digested fragments of WT COP1 and alternatively spliced variants.
