Fig. 4: DTX1 directs c-FLIPL into the endosome-lysosomal degradation pathway
From: c-FLIP is a target of the E3 ligase deltex1 in gastric cancer
a DTX1-induced c-FLIPL degradation was inhibited by NH4Cl and Leupeptin. DTX1-Myc and/or c-FLIPL-Flag were transfected into 293T cells. After 24 h, cells were untreated, or treated with MG132 (2.5 μM), NH4Cl (25 mM), or Leupeptin (100 μg/ml) for 8 h. The levels of c-FLIPL-Flag and DTX1-Myc were determined. b Mutation at the RING finger abolishes the ability of DTX1 to downregulate c-FLIPL. 293T cells were transfected with c-FLIPL-Flag with increasing amounts of HA-DTX1 or HA-DTX1-H2N2 (H453N, H456N). The levels of DTX1 and c-FLIPL were determined 24 h after transfection. c DTX1 directs c-FLIPL into endosome–lysosome compartments. 293T cells were transfected with mRFP-Rab5, LAMP1-mCherry, Cerulean-c-FLIPL, or EGFP-DTX1 as indicated. After 24 h, 293T cells were seeded onto polylysine-coated glass coverslips and allowed to attach for another 18 h. Cells were fixed and then mounted in DAPI-Fluoromount-G (Southern Biotechnology Associates). The images of EGFP, Cerulean, mRed, mCherry, and DAPI were obtained under a Zeiss LSM 780 confocal microscope (Zeiss). Experiments were independently repeated three times with similar results. Co-localization of Cerulean-c-FLIPL with mRFP-Rab5 or LAMP1-mCherry in 40–50 cells was calculated using WCIF ImageJ software. Scale bars, 10 μm, ***P ≤ 0.001
