Figure 2.

Viral protease 2A but not 3C cleaves overexpressed WT FLAG-DAP5 and endogenous DAP5 into 45- and 52-kDa truncates. (a) pIRES-2A or 3C plasmid was co-transfected into HeLa cells with WT FLAG-DAP5. As a control, HeLa cells were co-transfected with an empty pIRES vector and WT FLAG-DAP5 or with WT FLAG- DAP5 only. Cell lysates were collected 48 h after transfection and analyzed by western blot using an anti-FLAG antibody. β-actin was used as a loading control. (b) pIRES vector, pIRES-2A or pIRES 3C was transfected into HeLa cells. Lysates were collected for western blot analysis of endogenous 45-kDa DAP5-N. *DAP5 cleavage product (cp). Densitometric analysis was performed as indicated in Figure 1, with values presented under each blot. As a control, western blot was conducted on eIF4GI and eIF5B, to verify pIRES-2A and 3C constructs were effective at cleaving their respective substrates (c and d)