Extended Data Fig. 1: IGF1-induced AKT activation induces the translocation of PCK1 to the ER and the binding of PCK1 to INSIG1/2.

a, Huh7 cells with or without expression of Flag–INSIG1 (left) or Flag–INSIG2 (right) were treated with or without IGF1 (100 ng ml⁻¹) for 1 h. An immunoprecipitation assay was performed using anti-Flag antibody, and immunoprecipitates of Flag–INSIG1 or Flag–INSIG2 were eluted with Flag peptide, separated using SDS–PAGE and stained with Coomassie Brilliant Blue. Selected peptide hits of proteins associated with Flag–INSIG1 or Flag–INSIG2, identified through mass spectrometry, are shown. b, Hep3B cells were treated with or without IGF1 (100 ng ml⁻¹) for 1 h. c, Huh7 cells expressing Flag–PCK1 or Flag–PCK2 were stimulated with or without IGF1 (100 ng ml⁻¹) for 1 h. d, Huh7 cells expressing Flag–PCK1 or Flag–PCK2 were stimulated with or without IGF1 (100 ng ml⁻¹) for 1 h. Immunofluorescence analyses were performed with the indicated antibodies (top). The colocalization coefficients between the indicated proteins in the presence or absence of IGF1 are shown (bottom). At least n = 50 cells from each independent experiment were analysed and representative data are shown. Data are mean ± s.d. **P < 0.001 (two-tailed t-test). The regions in white boxes are shown at higher magnification on the right. e, Whole cell lysate (WCL), cytosolic and ER fractions were prepared from Huh7 and Hep3B cells stimulated with or without IGF1 (100 ng ml⁻¹) for 1 h. Cellular fractions from equal numbers of cells were analysed using immunoblotting with the indicated antibodies. f, Huh7 cells expressing Flag–PCK1 or Flag–PCK2 were stimulated with or without IGF1 (100 ng ml⁻¹) for 1 h. ER fractions and total lysates were prepared for immunoblotting analyses with the indicated antibodies. g, Total lysates were prepared from Huh7 cells pretreated with or without U0126 (20 μM), SP600125 (25 μM), SU6656 (4 μM) or MK-2206 (10 μM) for 30 min before treatment with or without IGF1 (100 ng ml⁻¹) for 1 h. h, Huh7 cells expressing wild-type HA–AKT1 or HA–AKT1-DN (K179A, T308A, S473A were treated with or without IGF1 (100 ng ml⁻¹) for 1 h. i, Huh7 cells were pretreated with or without U0126 (20 μM), SU6656 (4 μM), SP600125 (25 μM) or MK-2206 (10 μM) for 30 min before treatment with or without IGF1 (100 ng ml⁻¹) for 1 h. ER fractions and total lysates were isolated for immunoblotting analyses with the indicated antibodies. j, Huh7 cells were stably transfected with a control vector or a vector expressing HA–AKT1-DN (K179A, T308A, S473A). The cells were treated with or without IGF1 (100 ng ml⁻¹) for 1 h. ER fractions and total lysates were isolated for immunoblotting analyses with the indicated antibodies. k, Huh7 cells were pretreated with or without MK-2206 (10 μM) for 30 min and treated with or without IGF1 (100 ng ml⁻¹) for 1 h. Immunofluorescence analyses were performed with the indicated antibodies (left). The colocalization coefficients between the indicated proteins are shown (right). At least n = 50 cells from each independent experiment were analysed and representative data are shown. Data are mean ± s.d. **P < 0.001 (two-tailed t-test). l, Huh7 cells expressing Flag–PCK1 or Flag–PCK2 were transfected with or without HA–myr-AKT1. m, Huh7 and Hep3B cells were treated with or without IGF1 (100 ng ml⁻¹) for 1 h. In b, c, e–j, l, m, immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies, and the experiments were repeated three times independently with similar results.

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