Figure 1.

Identification of VDAC1 interacting sites in SOD1. (A) SH-SY5Y cells (4.5 × 10⁴ cells/well in 24-well plates) were transfected with an empty plasmid or a plasmid encoding for mutant SOD1^G93A or SOD1^G37R. Then, 24 h post-transfection, the cells were incubated for 5 h with (10-20)N-Ter-Antp peptide (20 µM) and then analyzed for cell death. The results are from triplicates of different biological repeats (n = 3) and are presented as the means ± SEM. * p < 0.05. (B) Schematic presentation of peptide arrays and detection of VDAC1-interacting peptides. (C) Glass-bound peptide array consisting of 768 overlapping peptides derived from 19 VDAC1-interacting proteins were incubated for 4 h with purified VDAC1 (64 nM) and then blotted with anti-VDAC1 antibodies against an internal sequence (1:5000) or with antibodies against the VDAC1-N-terminus, followed by incubation with HRP-conjugated anti-mouse IgG and detection using a chemiluminescence kit. Dark spots represent binding of VDAC1 to peptides derived from VDAC1-interacting proteins. Spots 1C16 and 1C17 (squared) represent SOD1-derived peptides interacting with VDAC1 as detected with anti-VDAC1 antibodies directed to an internal sequence, but not against the N-terminus. (D) SOD1 sequence with the two overlapping interacting peptides is indicated. (E) The 3D structure of the SOD1 (PDB_ID 1PU0) with the identified peptide localization indicated (in red ribbon). Images were prepared with UCSF-Chimera [50].