Over-expression of the chimeric YFP-HERV-K-T47D-RT enzyme. (A) CLSM analysis of 293T cells (left panel) and T47D cells (right panel) transiently or stably transfected, respectively, either with pYFP-C1 (row 1) or with pYFP-HERV-K-T47D-RT (row 2). Scale bars, 50 µm. (B) Western blot analysis of protein extracts obtained from 293T (left panel) and T47D cells (right panel) transfected with pYFP-C1, with wild type (WT) pYFP-HERV-K-T47D-RT, or with the mutated pYFP-HERV-K-T47D-RT. The blots were incubated with anti-GFP antibody. As a loading control, the same blots were incubated with anti-β-actin antibody. (C) RT-associated DNA polymerase colorimetric activity assay was performed on the same protein extracts as in panel (B), from either the 293T cells (left) or the T47D cells (right). The activity was normalized to the HERV-KT47D-RT-transfected cells' activity that was set to 100%. The RT assay was repeated three times, and this is a representative experiment.