FIG. 5.
v-Rel and c-Rel induce the expression of IRF-4 in transformed fibroblasts. (A) Expression of IRF-4 mRNA in uninfected fibroblasts (UN) and fibroblasts either infected with empty retroviral vector (DS3), transformed by DSv-Rel, infected with empty retroviral vector RCAS, or transformed by RCASc-Rel. Total RNA (10 μg) was subjected to Northern blot analysis with the probes described in Table 1. The viral genomic and spliced RNAs in DSv-Rel- or RCASc-Rel-infected cells shown in the panel labeled Rel were detected with a c-rel probe. The endogenous c-rel mRNA was below the threshold of detection at this exposure. The membrane hybridized with a c-rel probe was exposed to film for 2 h, while the IRF-4 membrane was exposed for 48 h. The intensity of the rRNA stained with ethidium bromide is shown in the bottom panel (rRNA). (B) Expression levels of IRF-4 and IRF-4ΔE6 mRNA in uninfected fibroblasts (UN) and fibroblasts transformed by DSv-Rel or transformed by RCASc-Rel were determined by RT-PCR. Lanes 1 to 3 show the products from these cell types after 35 cycles, while lane 4 shows the product from uninfected fibroblasts after 40 cycles. The PCR products corresponding to IRF-4 and IRF-4ΔE6 are indicated on the right side of the panel. (C) Western blot analysis of IRF-4 and Rel expression in fibroblasts. Whole-cell extracts from fibroblasts either infected with empty retroviral vector DS3, transformed by DSv-Rel, infected with CSV helper virus, or transformed by REV-TW and from sarcoma-derived fibroblastoid cell line 26T6 were analyzed. Lane 6 contains a mixture of lysate from CEFs overexpressing IRF4 from a retroviral vector and lysate from CSV-infected control CEFs (1:9). Extracts from 3 × 105 to 4 × 105 CEFs and 26TS cells were loaded per lane, blotted, and stained with anti-IRF-4 AI4-6 serum (upper panel) or anti-Rel HY87 antibody (lower panel). Positions of the two IRF-4 bands, a prominent background band (B), v-Rel, and c-Rel are indicated on the right side. The bands marked with the asterisk and numbers 1 to 4 in lane 5 are discussed in the text.