Figure 6

GILZ interacts with mTORC2. (a) mSin1 or scrambled (CTR) siRNA was transfected into M1 GILZ cells. One day post transfection, cells were lysed, and immunoprecipitation was performed using an anti-Rictor antibody, as described previously. (b) Rictor or scrambled (CTR) siRNA was transfected into M1 GILZ cells. One day post transfection, cells were lysed, and immunoprecipitation was performed using an anti-mTOR antibody, as described previously. (c) M1 GILZ cells were treated for 24 h with 100 nM rapamycin. Cell lysates (lower panel) and mTOR or Raptor immunoprecipitates (upper panels) obtained from cell lysates were analyzed for levels of the indicated proteins using WB. (d) In vitro kinase assay: mTORC2 was purified from M1 cells using an anti-Rictor antibody. mTORC2 kinase assays containing anti-GILZ or IgG immunoprecipitates were then performed using AKT as a substrate. (e) In vitro kinase assay: various concentrations of myc-tagged human recombinant GILZ were preincubated with immunoprecipitated Rictor, and recombinant, active human AKT1 was added.