Figure 2

Coexpression of Hsp90 enhances MLKL-mediated necroptosis. (a) The 293T cells were transfected with vector, with Flag-Hsp90α, with MLKL-Myc alone, or with Flag-Hsp90α and MLKL-Myc. After 24 h of transfection, cell images were taken with a Nikon-TE2000 microscope. Scale bar, 100 μm. (b) The 293T cells were transfected as in (a). After 24 h of transfection, cell death was quantified by propidium iodide (PI) staining. Cell death data are the means±S.D. of three independent experiments. (c) Hsp90α increases MLKL oligomerization. The 293T cells were transfected with the indicated plasmids. The cells were harvested 24 h after transfection, and non-reducing samples (without β-mercaptoethanol) of whole-cell lysate were analyzed by immunoblotting with anti-HA antibody. (d) Hsp90α increases the plasma membrane translocation of MLKL. The 293T cells were transfected with the indicated expression vectors. After 24 h of transfection, the cells were harvested and lysed in Triton X-114 lysis buffer and then separated into aqueous phase (Aq) and detergent phase (Det) as described in the experimental procedures. The samples were resolved and probed with the indicated antibodies. β-actin and Cox4 were used as loading controls for soluble protein and membrane protein, respectively