Figure 1
From: Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis

Hsp90 interacts with MLKL and RIP3. (a) Hsp90α interacts with MLKL in a yeast two-hybrid assay. AH109 yeast cells were co-transformed with the pACT2-Hsp90α (642–732aa) and pGBKT7-MLKL (1–178aa) plasmids and then plated on SD/-Leu/-Trp, SD/-His/-Leu/-Trp or SD/-Ade/-His/-Leu/-Trp selection media to allow growing for 1 week. Positive control pGBKT7-p53+pGADT7-T, negative control pGBKT7-Lam+pGADT7-T. (b) Flag-Hsp90α interacts with Myc-MLKL in vivo. The HEK293T cells were co-transfected with Flag-Hsp90α and Myc-MLKL, and cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with anti-Flag or anti-Myc antibody. (c) Myc-MLKL binds to Flag-Hsp90α. The 293T cells were co-transfected with Myc-MLKL and Flag- Hsp90α, and whole-cell lysates were immunoprecipitated with the indicated antibodies and subsequently analyzed by western blotting for the indicated proteins. (d) Endogenous Hsp90 associates with MLKL. Human Hela cervical carcinoma cell line whole-cell lysates were immunoprecipitated with the indicated antibodies and subsequently probed with anti-Hsp90 or anti-MLKL antibody. The asterisk denotes nonspecific IgG band. (e) Mapping of the MLKL domains responsible for binding to Hsp90α. Myc-MLKL or the indicated Myc-MLKL fragments and Myc-GFP were co-transfected with Flag-Hsp90α into 293T cells. After 24 h of transfection, cell lysates were immunoprecipitated with the indicated antibodies before western blotting. The asterisks denote nonspecific IgG bands. (f) Schematic representations of WT MLKL and the deletion mutants of MLKL used in the experiment. (g) Flag-RIP3 associates with Myc-Hsp90α in vivo. The 293T cells were co-transfected with Flag-RIP3 and Myc-Hsp90α, and cell lysates were immunoprecipitated with the indicated antibodies and probed with anti-Myc or anti-Flag antibody