Expression level of c-Myb in T2ECs expressing v-ErbA or the S61G form of v-ErbA. 3A – Effect of v-ErbA on the expression level of the c-myb gene. Total RNA was extracted from T2ECs expressing either the oncogenic form or the non-transforming form of v-erbA. A reverse transcription and real-time PCR analysis were performed to quantify the expression level of c-myb gene. The fold variation is represented as VA/NTVA ratio and corresponds to a decrease or an increase of the c-myb mRNA in T2ECs expressing the transforming form of v-ErbA (VA) in comparison with T2ECs expressing the non-transforming form of v-ErbA (NTVA). The grey bars represent a mean of ratio calculated using the three reference genes T-Complex 1, hnRNP and ATP synthase subunit B1. The hatched bar is the mean of mRNA accumulation in the five independent experiments. 3B – Effect of v-ErbA on the expression level of the c-Myb protein. T2ECs were either left non-infected (T2ECs), infected by a retrovirus carrying the wild type v-ErbA (VA) or carrying the point-mutated form of v-ErbA, S61G (NTVA). Protein extracts (30 μg) were analyzed and two separate western blots were probed with an anti-c-Myb or anti-GAPDH antibody. 3C – Different effects of v-ErbA and S61G on c-Myb responsive transcription. T2ECs were transfected with reporter plasmids containing either five wild-type (EW5) or mutant (EM5) Myb-binding sites upstream of a simple TATA box. Each reporter was tested with a vector expressing or not the transforming form of v-ErbA (VA) or a vector expressing the S61G mutant for v-ErbA (NTVA) and the β-Galactosidase expressing plasmid. Twenty-four hours after transfection, the cells were analyzed for luciferase activity, which reflects the ability of the c-Myb protein to regulate the gene expression. Luciferase activities were normalized by using β-Gal expression as an internal control. Luciferase activity in the presence of EW5 and the transforming form of v-ErbA was assigned to a value of 100%. The pGL2 plasmid transfected in normal T2ECs represents the negative control (vector which does not contain any binding sites upstream the luciferase coding region). Data are average of two (EM5 and pGL2) to three experiments (EW5) and error bars indicate maximum and minimum values of different sets of data points.