Fig. 2

CCT contributes to mTORC assembly. a Work flow of the CCT depletion experiments. Bold letters correspond to the figure panels in which the experimental results are displayed. b Effects of CCT depletion on endogenous mTORC subunit expression. Cells were treated with CCT5 sgRNA or control sgRNA and Cas9, lysates were immunoblotted and band intensities were quantified as indicated. Data are shown as a percent of the control. Bars represent the average ± standard error. c Effects of CCT depletion on co-immunoprecipitation of mTORC1 subunits. Cells were CRISPR treated and transfected with the indicated mTORC1 subunits and a GFP control. mTOR immunoprecipitates were immunoblotted and quantified as indicated. d Lysates from cells in panel (c) were immunoblotted and quantified for expression of mTORC1 subunits and controls as indicated. e Effects of CCT depletion on co-immunoprecipitation of mTORC2 subunits with mTOR. Cells were CRISPR treated and transfected with the indicated mTORC2 components and a GFP control. mTOR immunoprecipitates were immunoblotted and quantified as indicated. f Lysates from the cells in panel (e) were immunoblotted and quantified for expression of mTORC2 subunits and controls as indicated. g Effects of CCT depletion on co-immunoprecipitation of Gγ with Gβ. Cells were CRISPR treated and transfected with Gβ, Gγ, and a GFP control. Gβ immunoprecipitates were immunoblotted and quantified as indicated. h Lysates from the cells in panel g were immunoblotted and quantified for expression of Gβ and Gγ and controls as indicated. i Effects of siRNA-mediated CCT depletion on insulin-mediated IRS1 S636/639 phosphorylation or AKT S473 phosphorylation in HEPG2 cells. Cells were treated with siRNAs to CCT1/CCT5, mLST8, Raptor or a non-targeting control, serum starved for 18 h and then treated with insulin. Cell lysates were immunoblotted as indicated. *p < 0.05, **p < 0.01, ***p < 0.005. Source data are provided in the Source Data file