Characterization of DsRed-NT5E fusion proteins.

<p>A. COS-7 cells were transiently transfected with an expression vector for DsRed fused to wild type form of human NT5E (DsRed-hNT5E WT) and fusion proteins were localized by immunofluorescence with antibodies against fluorescent DsRed (green) and native hNT5E (red) proteins, and by internal fluorescence (purple). The merged picture shows obvious overlap of all 3 signals indicating that DsRed-hNT5E fusion protein is intact and contains both antigens. <i>Magnification 400X</i>. B. Immunoblot analysis of total protein extracts from mock-transfected (mock) or transfected COS-7 cells with expression vectors for DsRed monomer and DsRed-hNT5E (DsRed NT5E) fusion protein using DsRed (left) and hNT5E (right) antibodies. Both antibodies detect a protein with a molecular weight of approximately 90 kiloDaltons (black arrowheads, left and right) while DsRed antibody only detects the DsRed monomer (white arrowhead, left) indicating that DsRed-hNT5E fusion is intact and contains both antigens. Total β-actin protein was used as loading control. C. Enzyme histochemistry of mock-transfected or transfected COS-7 cells with expression vectors for DsRed-hNT5E wild type (WT) and mutant (F1–3) proteins using adenosine monophosphate (AMP) as substrate. Only COS–7 cells transfected with the expression vector for DsRed-hNT5E wild type fusion protein displayed AMPase activity, which could be blocked in presence of known NT5E inhibitor α/β-methylene-ADP. Mock-transfected or COS-7 cells transfected with expression vectors for DsRed-hNT5E mutant (F1–3) proteins lack AMPase activity. <i>Magnification 200X</i>.