Expression of ALS analogs. HepG2 cells were infected with vector (vec; lanes 2 and 3), recombinant wild-type ALS (wtALS; lanes 4 and 5), D440N-ALS (lanes 6 and 7), D440N/T442A-ALS (lanes 8–10), or T442A-ALS (lanes 11–13) adenoviruses for 24 h. Samples of culture media (panel A) or cell lysates (panel B) were separated on SDS-PAGE and immunoblotted for ALS. Samples of cell lysates were also immunoblotted for GFP as a marker of infection and protein expression efficiency (panel C). As a control, 10 ng of purified recombinant wild-type ALS protein (rec) was loaded in lane 1. D, Densitometric scan of the immunoblots above. ALS levels are normalized to GFP levels and expressed as the ratio of intracellular/secreted levels. The data shown are mean ± sd expressed relative to recombinant wild-type ALS from at least two separate experiments. *, P ≤ 0.0001 compared with wild-type ALS.