Analysis of the Sox4 retroviral vector. (A) Schematic of the MIGR1-based retroviral vectors. The complete cDNA of Sox4 (light gray rectangle) including a C-terminal V5 epitope tag (dark gray square) was inserted into the MIGR1 vector. The MIGR1 and the MIGR1-p185 vectors were gifts of Warren S. Pear. The MIGR1-p185 vector contains the complete Bcr-abl cDNA (dotted rectangle). All vectors contain an IRES (internal ribosome entry site) and the EGFP cDNA. The arrow depicts the start of transcription. (B) Results of transient cotransfection assays. NIH 3T3 cells were transiently cotransfected with either 5 μg MIGR1 or MIGR1-Sox4 expression plasmid (as indicated on the x-axis) and 1 μg of either reporter plasmid, pTATAluc (▪) or pTATAluc Sx3X (□). The pTATAluc Sx3X contains 3 copies of the SOX4 consensus binding site. Cells were harvested 48 hours following FuGene transfection, and lysates from an equal number of cells were analyzed for relative luciferase units (RLUs). The results are presented as the RLU value of cells transfected with both reporter and expression plasmids divided by cells transfected by reporter alone. Results are presented as an average of 2 experiments with the standard error indicated. (C) Western blot of 32Dwt18 clones. Single-cell-derived 32Dwt18 clones expressing GFP were isolated and expanded into clonal cell lines (Sox4 lines A-C, MIGR1 lines A-B, and p185 lines A-B). Nuclear extracts were isolated from an equal number of cells from each cell line. Equal volumes of extract were separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. Membranes were sequentially probed with anti-V5 and anti-abl antibodies. The anti-V5 antibody detects an approximately 47-kDa band in the Sox4 clones (top row), and the anti-abl antibody detects an approximately 185-kDa band in the p185 clones.