Establishment of MlFosER cell lines that express deregulated c-Myc (MlFosER-myc). (A) MlFosER-myc and M1myc cells were established by infecting MlFosER and M1 cells, respectively, with the MSCV-myc/puro retroviral vector, followed by selection of cells in G418 + puro or puro as described in “Materials and methods.” Indicated clones were tested for c-Myc and c-FosER RNA expression using RNA blots. RNA (10 μg/lane) was resolved on a 1% agarose formaldehyde gel and transferred to nylon membranes (Duralon) for Northern blot analysis. Blots were hybridized with ³²P-labeled c-Myc probe, then stripped and rehybridized with a c-fos probe (to detect c-fosER RNA; none of the clones expressed endogenous c-fos) as described in “Materials and methods.” (B) Western blot to test for FosER expression. Protein lysates were prepared from indicated cell lines, and 50 μg/well was run on a 10% SDS-PAGE gel. The gels were transferred onto a PVDF membrane (Millipore) and probed with a primary antibody specific for c-Fos (Santa Cruz Biotechnology). Detection of signal was done as described in “Materials and methods.”