PRAS40 and PRR5-Like Protein Are New mTOR Interactors that Regulate Apoptosis (original) (raw)
Figure 4
PRAS40 and PRR5L are pro-apoptotic.
A. PRR5L and PRAS40 knock down cells are resistant to TNFα/cycloheximide induced apoptosis. HeLa cells were transfected with PRR5L, PRAS40 or control siRNA and incubated for 48 h, followed by 2 h induction of apoptosis with TNFα and cycloheximide. Cells were fixed and stained with DAPI and cleaved PARP antibody, and the percentage of apoptotic cells was quantified. B. PRAS40's effect on apoptosis is independent of mTORC1. HeLa cells were transfected with PRAS40 or control siRNA and incubated for 48 h, and treated with 100 nM rapamycin or carrier for 1 h before incubation with TNFα and cycloheximide for 2 h to induce apoptosis. Extracts were analyzed by immunoblotting with the indicated antibodies. C. PRR5L deficiency protects against apoptosis in SIN1 deficient cells. HeLa cells were transfected with diced PRR5L siRNA and/or synthetic siRNA against SIN1 as indicated, or the appropriate control siRNAs. Cells were incubated for 48 h, and apoptosis was induced with TNFα and cycloheximide for 2 h. Cells were fixed and stained with DAPI and cleaved PARP antibody, and the percentage of apoptotic cells was quantified. The efficiency of SIN1 knock down was assessed in parallel by immunoblotting (right panel).