Figure 5
From: AMPKα2 regulates fasting-induced hyperketonemia by suppressing SCOT ubiquitination and degradation
AMPKα2 physiologically binds and stabilizes SCOT. (A) Representative western blot images of Myc-AMPKα2 and SCOT in HEK293T cells. HEK293T cells were transfected with Myc or Myc-AMPKα2 for 36 h; Myc-trap agarose was used to immunoprecipate the Myc-tagged protein. (B) Representative western blot images of endogenous AMPKα2 and SCOT protein binding of C2C12 cells. C2C12 were cultured to confluent and differentiation for 48 h. Then treated with Compound C (10 µM) for 6 h. The cell lysate in IP buffer and assayed the protein concentration with BCA kit. Total 2 mg proteins for each IP with rabbit IgG or anti-SCOT antibody with Goat anti-rabbit IgG (NEB, S1432) O/N in 4°. The beads were washed and resulted solution for WB with Rabbit true blot anti-rabbit-IgG-HRP (Rockland, 18-8816-31). (C) Representative western blot images of Myc-AMPKα2 and SCOT protein. The HEK293 cells were transfected with Myc or Myc-AMPKα2 for 36 h and treated with/out Compound C (10 µM) for 6 h. The cell lysate in IP buffer and assayed the protein concentration with BCA kit. Total 2 mg proteins for each IP with Myc-trap agarose O/N in 4°. The beads were washed and resulted solution for WB with Rabbit true blot anti-rabbit-IgG-HRP (Rockland, 18-8816-31) (D) Representative western blot images of AMPKα2 fragments and GST-SCOT in HEK293T cells. HEK293T cells were transfected with MYC-AMPKα2 (domains 1, 2, or 3) and GST or GST-SCOT for 36 h and then glutathione Sepharose beads were used to pull down the GST-SCOT protein. (E) Representative western blot images of AMPKα2 domain 1 and GST-SCOT in HEK293T cells. HEK293T cells were transfected with MYC-AMPKα2 domain 1 and GST-SCOT for 36 h and treated with 10 µM Compound C for 6 h. Next, glutathione Sepharose beads were used to pull down the GST-SCOT protein. (F) Representative western blot images of AMPKα2 WT, KD AMPKα2, and GST-SCOT in HEK293T cells. HEK293T cells were transfected with MYC-AMPKα2 WT and MYC-KD AMPKα2 for 36 h; glutathione Sepharose beads were then used to pull down the GST-SCOT protein. (G) Representative western blot images of ubiquitinated SCOT in HEK293T cells transfected with GST-SCOT, GFP-UB, and MYC-AMPKα2 WT (or KD MYC-AMPKα2) for 36 h; glutathione Sepharose beads were then used to pull down the GST-SCOT protein.
