Figure 5
From: DJ-1 maintains energy and glucose homeostasis by regulating the function of brown adipose tissue
DJ-1 promotes PTEN degradation through E3 ligase Mib2. (a) Lysates of BAT were immunoprecipitated with DJ-1 antibody followed by immunoblotting with Mib2 antibody. (b) Endogenous co-IP assay of Mib2 and PTEN was performed in wild-type or DJ-1 KO brown adipocytes. (c) The ubiquitination of Mib2 was examined in HEK293T cells after transfected with GFP-tagged Mib2, HA-tagged ubiquitin and Myc-tagged DJ-1 or their control vectors. After 16 h transfected, the cells were treated with MG132 for 8 h. Cell lysates were immunoprecipitated with GFP antibody and immunoblotted with HA antibody. (d) The ubiquitination of PTEN was examined in HEK293T cells after transfected with the indicated plasmids. Cells were treated and analysis as in c. (e) Mib2 ubiquitination PTEN in vitro. His-PTEN was incubated with ubiquitin, E1, E2 (Ubc4) and ubiquitin ligase (Mib2, CHIP, Parkin), as indicated, and then it was analyzed by immunoblot using anti-His antibody. (f, g) Endogenous PTEN ubiqutination was examined in brown adipocytes with Mib2 overexpression (f) or Mib2 knockdown (g). Cells were treated with MG132 for 8 h. Cell lysates were immunoprecipitated with PTEN antibody and immunoblotted with ubiquitin antibody. (h) HEK293T cells were transfected with Flag-tagged WT Mib2 or RING domain truncation (ΔRING) Mib2 plasmids alone or together with HA-tagged ubiquitin, GFP-tagged PTEN. The cells then treated and analysis as in (c). (i) DJ-1 promotes Mib2 ubiquitination PTEN in vitro. His-PTEN was incubated with ubiquitin, E1, E2 (Ubc4), GST-DJ-1 and ubiquitin ligase (Mib2, CHIP and Parkin), as indicated, and then it was analyzed by immunoblot using anti-His antibody. GST-DJ-1 increased PTEN ubiqutination to 1.3-fold as indicated in the bottom of the panel. (j) Endogenous PTEN ubiqutination was examined in brown adipocytes with DJ-1-overexpression and/or Mib2 knockdown. Cells treated and analysis as in f. (k) Lysates of brown adipocytes with Mib2 overexpression or knockdown were immunoblotted with the indicated antibodies. (l) Ucp1-luciferase activity was analyzed in brown adipocytes with Mib2 overexpression or knockdown.
