Fig 1: Inhibition of mTORC1 and cell death sensitivity caused by raptor processing and decrease of mTOR-raptor interaction. (a) Jurkat T cells were treated with STS or FasL for 2, 4 or 6 h. Raptor processing, phosphorylation of mTORC1 substrates (S6K, 4E-BP1), caspase-3 and -6 activations were analyzed. (b) Untreated or STS-treated Jurkat T-cell lysates were IP with an antibody raised against mTOR. Raptor–mTOR interaction, PARP and raptor cleavage were analyzed. (c) Jurkat T cells were transfected with WT or DDAAA raptor constructs and treated with STS for 4 h. A live/dead staining was used to quantify the percentage of cell death by flow cytometry. The unpaired Student’s t-test (Prism software) was used to calculate the significative difference. **P=0.0025. Data are represented as mean±S.E.M.; n=3.
Fig 2: Raptor cleavage by caspase-6 and other caspases. (a) Jurkat T-cell lysates were incubated with recombinant caspases-3, -6, -7 for 2 h. Cleavage of raptor and physiological substrates of caspases (PARP and lamin A/C) were monitored and compared with the positive control sample (Jurkat T cells treated with STS for 2 h). (b) The effect of z-VAD-fmk on the recombinant caspase-6-mediated cleavage of raptor in Jurkat T-cell lysate. (c) Cartoon of recombinant mTORC1 (composed of mLST8, mTOR (1362-end) and recombinant raptor). FAT, focal adhesion targeting; FRB, FKBP12-rapamycin binding; FATC, C-term focal adhesion targeting. (d) Cleavage of the full-length recombinant raptor (inside recombinant mTORC1) by recombinant caspase-6, -3 and -7 in the presence (or not) of z-VAD-fmk. (e) WT versus caspase-6 K.O. KBM-7 cells were treated with STS or FasL for 4 h and then cell lysates were analyzed by SDS-PAGE and western blot.
Fig 3: Raptor cleavage upon drug treatment in various T lymphoma and B-cell lines. Lymphoma T (Jurkat, Hut78) or B-cell lines (HBL-1, BJAB, SUDHL4) were incubated with 50 μM of cisplatin for 15 h (a), 50 μM of etoposide for 15 h (b), 25 μM of curcumin for 15 h (c), 1 μM of rapamycin for 16 or 48 h (d), 50ng/ml of FasL for 4 or 8 h (e and f) and with 2 μM of STS for 2, 4, 6, 8 or 24 h (g and h). Cell lysates were then analyzed on 8% SDS-PAGE. (i) Scheme of raptor protein highlighted with the region recognized by the raptor mAb and with the putative cleavage site. RNC, raptor N-term conserved domain; HEAT, Huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A), and the yeast kinase TOR1; WD, tryptophan-aspartic acid (WD) dipeptide.
Supplier Page from Abcam for Recombinant Human Raptor protein (GST tag N-Terminus)