Fig. 3

Fc-mediated intracellular delivery of copper/zinc-superoxide dismutase (SOD1) in macrophages. Cells loading (2 × 10⁶ cells/ml) were plated in 24-well culture plates, and incubated overnight in the presence or absence of the immune complexes (IgG1 IC or SOD1 IC) and/or free SOD1. As already described in Materials and Methods for analysis of Bo-SOD1 loading the cells were then treated as already described with 0.5% trypsin for 5 min at 37°C to digest residual SOD, which may be bound to the external surface of the cell membrane. After neutralization of the trypsin, the cells were washed and protein was extracted with a buffer containing 0.1 M Na₂CO₃ (pH 10.2), 0.2 mM EDTA, 0.2% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The soluble extracts were then assayed by specific ELISA for the presence of bovine SOD1 and data represent the mean ± SEM of four different experiments.