C/EBP retroviral constructs.

(A) Expression vectors containing a bicistronic system expressing the gene of interest and GFP under the control of the MSCV promoter. MSCV-IRES-GFP (MIG) is an expression vector with a multiple cloning site (MCS), at which either a human C/EBPα (42 kDa) or human C/EBPε (32 kDa) cDNA was inserted. The human C/EBPα includes an open reading frame (uORF) upstream to the start codon for C/EBPα. rC/EBPα-WT is similar to the hC/EBPα construct, but contains rat C/EBPα. rC/EBPα-ΔuORF is rat C/EBPα without the upstream open reading frame (uORF). The hC/EBPε-ER^TM retroviral construct contains human C/EBPε fused at the C-terminus to a tamoxifen-responsive mouse estrogen receptor hormone-binding domain. (B) Western blots of whole-cell lysates of transduced FDC-P1 cells. Lysates were prepared at 24 hours after the second round of transduction. (i) FDC-P1 cells were transduced with retroviruses expressing MIG (control), hC/EBPα, rC/EBPα-WT, and rC/EBPα-ΔuORF. Lysates were blotted with rabbit polyclonal antibodies raised to an internal region of rat C/EBPα. Arrow indicates the position of C/EBPα. (ii) Lysates of MIG-, hC/EBPε-, and hC/EBPε-ER^TM–transduced FDC-P1 cells were blotted with rabbit polyclonal antibodies raised to the carboxy terminus of rat C/EBPε. Arrows indicate the positions of hC/EBPε (32 kDa) and hC/EBPε-ER^TM (70 kDa). The identities of the cross-reacting bands in the hC/EBPε lane are uncertain. The bands in the hC/EBPε-ER^TM lane below the full-length hC/EBPε-ER^TM may represent smaller hC/EBPε-ER translation products initiated from downstream AUG codons.43