Figure 1.

TNPO3 depletion and rescue with non-targetable TNPO3 cDNA. (A) Schematic representation of the lentiviral vectors used to generate TNPO3 knockdown (KD) and rescue cell lines. (B) The sequence of steps used to obtain the four pools of stable cell lines. HeLa cells were transduced with control (Ctrl) KD vector or with TNPO3 KD vector, and selected in pools with 10 μg/ml of puromycin. Each pool of KD cells was then transduced a second time with the rescue vector, either empty or bearing non-targetable TNPO3 cDNA (ntTNPO3), and selected in pools with 10 μg/ml of blasticidin, as well as 1 μg/ml puromycin. (C) Steady-state levels of TNPO3 protein in each of the four pools of doubly-tranduced cells. Cell lysate was probed in western blots with anti-TNPO3 antibody (upper panel) and anti-β-actin antibody (lower panel).