Figure 3 | Cell Death & Disease

Figure 3

From: Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis

Figure 3

Hsp90 regulates MLKL and RIP3 stability. (a) 17AAG decreases the levels of ectopically expressed Myc-MLKL protein. HeLa cells were transfected with the Myc-MLKL plasmid. After 24 h of transfection, cells were treated with 250 nM 17AAG for the indicated times. Myc-MLKL protein levels were analyzed by western blotting with anti-Myc antibody. (b) HeLa cells transfected with Myc-MLKL were treated with the indicated concentrations of 17AAG for 24 h. Cell lysates were probed with anti-Myc antibody. (c) 17AAG reduces endogenous MLKL protein levels. HeLa cells were treated with 250 nM 17AAG for the indicated times. The cell lysates were resolved on SDS-PAGE and analyzed by immunoblotting with anti-MLKL-specific antibody. Actin was used as a loading control. (d) HeLa cells were treated with the indicated concentrations of 17AAG for 24 h. The levels of endogenous MLKL protein were analyzed by western blotting with anti-MLKL-specific antibody. Actin was the loading control. (e) 17AAG-induced degradation of MLKL is mediated by the proteasome pathway. HeLa cells transfected with Myc-MLKL were treated with or without 250 nM 17AAG in the presence or absence of 25 μM MG132 for 8 h as indicated. MG132 was added 0.5 h prior to 17AAG treatment. Cells were lysed with Triton X-100 lysis buffer (1% Triton X-100). Insoluble precipitates were resolubilized with 2% SDS lysis buffer. Equal amounts of protein were analyzed by western blotting with anti-Myc antibody. (f) 17AAG affects exogenous RIP3 protein levels. HeLa cells transfected with HA-RIP3 were treated with increasing concentrations of 17AAG for 24 h. The levels of HA-RIP3 were determined by western blotting with anti-HA antibody. (g) The effect of 17AAG on endogenous RIP1/RIP3/MLKL protein levels. HT29 cells were treated with different concentrations of 17AAG for 24 h. Cell lysates were probed with anti-RIP1, anti-RIP3 and anti-MLKL-specific antibodies. Actin was the loading control. (h, j) 17AAG decreases the half-life of endogenous MLKL protein. HeLa cells were treated with 250 nM 17AAG for 12 h before the addition of CHX (25 μg/ml). Cells were harvested at the indicated times after CHX addition, and the levels of MLKL protein were determined by immunoblotting with anti-MLKL-specific antibody. (i, k) Quantification of the experiments shown in (h) and (j). The values shown are obtained from three independent experiments and are normalized to the β-actin control

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