c-myb induces endogenous cyclin A1 expression in human embryonic fibroblasts (HEF). Primary human embryonic fibroblasts were transfected, and after 48 hours, they were sorted by flow cytometry using EGFP expression as a marker of transfection (see Materials and Methods). HEF were transfected either with empty vector alone (HEF + vector) or with a c-myb expression vector (HEF + c-myb). Nontransfected cells of the latter transfection were also sorted and served as an additional control (HEF control). Finally, a sample with ddH2O instead of RNA for the reverse transcription reaction served as a negative control (negative control). After the PCR reactions for cyclin A1 (28 cycles, 382 bp) and β-actin (20 cycles, 210 bp), products were run on agarose gels, blotted, and hybridized with digoxigenin-labeled internal oligonucleotides. Detection was performed using antidigoxigenin antibody and a chemiluminescence reaction. The digoxigenin-labeled marker VI (Boehringer Mannheim) was run in parallel: 154, 220/234, 298, 394, 453, 517, 653, and 1,033 bp. The 154-bp band of the marker is too weak to be seen on the β-actin blot.
