Figure 5

Lysine 36 of PARP-2 is necessary for the binding of PARP-2 to importin α3. A) Importins α1, α3, α5 and α7 were expressed as GST-fusion proteins in E. coli and purified with Glutathione Sepharose 4B beads. Expression was checked by SDS-PAGE followed by Coomassie staining. B) PARP-2 binds mostly to importin α3 and to a lower extent to importin α1 and α5. Purified GST-importins were incubated with whole cell extracts from HEK293T cells, either untransfected or transfected with wild type HA-PARP-2, then western blot analysis was performed using an anti-HA antibody. C) Lysines 36/37 are required for the binding of PARP-2 to importin α3. Purified GST-importin α3 was incubated with whole cell extracts from HEK293T cells transfected with either wild type (wt) HA-PARP-2 or with the indicated double and quadruple mutants. Proteins were separated by SDS-PAGE and analyzed by western blot using an anti-HA antibody. D) Lysine 36 but not lysine 37 is required for the binding of PARP-2 to importin α3. GST-importin α3 was bound to Glutathione Sepharose 4B and incubated with whole cell extracts from HEK293T cells expressing either wild type (wt) PARP-2 or the indicated single mutants. PARP-2 bound to importin α3 was detected using an anti-HA antibody.