Figure 1.

Overexpression of G392E-mutant neuroserpin in HEK-293 cells does not trigger cytotoxicity. HEK-293 cells were stably transfected with plasmids encoding human wild-type or G392E-mutant neuroserpin. Cell extracts (cell) and culture media (medium) were analyzed by SDS-PAGE (a) and non-denaturing PAGE (b) followed by western blot analysis with an antibody directed against neuroserpin. β-actin was used as loading control. The black arrow identifies the 50 kDa main form of neuroserpin, the red arrow a partially glycosylated form, the blue arrow the unglycosylated protein, and the green arrow G392E-mutant neuroserpin carrying an extra glycan chain on the asparagine residue 401. Cells stably transfected with empty vector were used as negative control (neg). Expression of the mutant form of neuroserpin did not lead to cell toxicity in HEK-293 cells, as shown in these cultures by quantification of LDH activity in the culture media (c, LDH assay) and cell proliferation (d, MTT assay). Western blot membranes shown in (a) and (b) have been cropped, full-length blots are presented in Supplementary Fig. S3. Values are mean + /− SD; n = 3; p = 0.5583 for LDH assay; p = 0.1029 for MTT assay.