To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the a 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. Cells were then cultured for 2 weeks under selective pressure in the presence of 500 mg/ml Hygromycin B, the selection agent for the a 1 -antitrypsin expressing plasmid, and 500 mg/ml G418, the selection agent for the rtTA (reverse Tet-controlled transactivator) cassette. The selective media were replaced every 3 days. Well-separated antibiotic-resistant cell clones were individually isolated with cloning cylinders (Sigma). Therefore, a small volume of Trypsin-EDTA (Sigma) was added and the culture dish was incubated briefly at 37uC until cells detach. Cells were then collected from inside the cylinder and transferred to individual wells of a 24-well plate for further growth in selective medium. When grown to confluence, cells were transferred to larger flasks. Protein expression was induced with 1 mg/ml doxycycline (Clontech) and analyzed by Western Blot and immunofluorescence. Stable cell lines showing similar expression levels of the various a 1 -antitrypsins were chosen for further experiments.