Fig. 3

HIV-1 PR cleaves endogenous RIPK1. a HEK293T cells were transfected with empty vector (as control) or increasing amounts of an expression plasmid encoding catalytically active HIV-1 PR. Addition of SQV (5 μM) or transfection of catalytically inactive HIV PR D25 N served as negative controls. Cells were lysed 24 h after transfection and total cell extracts were subjected to SDS-PAGE and WB. Proteins were revealed using antibodies against RIKP1 (rabbit monoclonal antibody from Cell Signaling), β-actin, or HIV-1 PR. b HIV-1 PR cleaves endogenous RIPK1 in vitro. Jurkat cell lysates were incubated with recombinant HIV-1 PR (at a ratio of 1000:1) in the absence or presence of SQV (5 μM). After 3 h of incubation at 37 °C, lysates were subjected to SDS-PAGE and Western blotting (WB). Proteins were revealed using antibodies described above. c Jurkat cells with stably integrated Tet-inducible vector encoding catalytically active HIV-1 PR were cultured without (-DOX) or with doxycycline (+DOX, 1 μg/ml) in the absence or presence of SQV (5 μM). Total cell lysates were prepared 24 h after treatment and subjected to SDS-PAGE and WB. Proteins were revealed using antibodies as described above