Extended Data Fig. 4: Generation of Huh7 and Hep3B cells with knock-in expression of both INSIG1(S207A) and INSIG2(S151A), and PCK1-mediated phosphorylation of INSIG1/2 reduces the binding of oxysterols to INSIG1/2.

a, b, Genomic DNA was extracted from two individual clones of parental Huh7 and Hep3B cells with knock-in expression of INSIG1(S207A) (a) and INSIG2(S151A) (b). PCR products amplified from the indicated DNA fragments were separated on an agarose gel. c, d, Sequencing of parental and two individual clones of parental Huh7 and Hep3B cells with knock-in expression of INSIG1(S207A)/INSIG2(S151A) double mutants. The red line indicates the sgRNA-targeting sequence. The black line indicates the PAM. Blue arrows indicate mutated nucleotides. A mutated amino acid and its wild-type counterpart are indicated by the solid red box. e, Top, Flag/His-tagged INSIG2 immunoprecipitated and purified from Huh7 cells was incubated with the indicated GST–PCK1 proteins with or without active GST–AKT1 in the presence of ATP and GTP for 1 h. Immunoblotting analyses were performed with the indicated antibodies. Bottom, INSIG2-conjugated Ni-NTA agarose beads were washed and incubated with 400 nM [³H]25-hydroxycholesterol. Specifically bound [³H]25-hydroxycholesterol was measured (n = 6). **P < 0.001 (two-tailed t-test); NS, not significant (P=0.875, 0.846, 0.969, 0.924 (left to right)). f, g, Top, Flag/His-tagged wild-type INSIG1 or INSIG1(S207A) (f) or Flag/His-tagged wild-type INSIG2 or INSIG2(S151A) (g) purified from Huh7 cells were incubated with purified wild-type GST–PCK1, GST–PCK1(S90D) or GST–PCK1(S90E) in the presence of GTP for 1 h. Immunoblotting analyses were performed with the indicated antibodies. Bottom, the INSIG1 or INSIG2 proteins on Ni-NTA agarose beads were washed with PBS five times and incubated with 400 nM [³H]25-hydroxycholesterol. Specifically bound [³H]25-hydroxycholesterol was measured (n = 6). **P < 0.001 (two-tailed t-test); NS, not significant (P=0.823, 0.445, 0.185 (left to right) (f); P=0.320, 0.196, 0.735 (left to right) (g)). h, Top, Flag/His-tagged wild-type INSIG2 or INSIG2(S151A) purified from Huh7 cells were incubated with purified wild-type GST–PCK1 with or without purified active GST–AKT1 in the presence of ATP and GTP for 1 h. Immunoblotting analyses were performed with the indicated antibodies. Bottom, the INSIG2 protein on Ni-NTA agarose beads was washed with PBS five times and incubated with 400 nM [³H]25-hydroxycholesterol. Specifically bound [³H]25-hydroxycholesterol was measured (n = 6). **P < 0.001 (two-tailed t-test); NS, not significant (P = 0.682, 0.947 (left to right)). i, j, Flag/His-tagged wild-type INSIG1 or INSIG1(S207A) (i) or wild-type INSIG2 or INSIG2(S151A) (j) were purified from Huh7 treated with or without IGF1 (100 ng ml⁻¹) for 12 h. Immunoblotting analyses were performed with the indicated antibodies (left). The INSIG1 or INSIG2 proteins on Ni-NTA agarose beads were incubated with the indicated concentration of [³H]25-hydroxycholesterol. Specifically bound [³H]25-hydroxycholesterol was measured (n = 6) (right). k, l, Top, Flag/His-tagged INSIG1 (k) or INSIG2 (l) was expressed in parental Huh7 cells and the Huh7 cells with knock-in expression of PCK1(S90A). After these cells were treated with or without IGF1 (100 ng ml⁻¹) for 12 h, immunoblotting analyses were performed with the indicated antibodies. Bottom, the immunoprecipitated and purified INSIG1 or INSIG2 was incubated with the indicated concentration of [³H]25-hydroxycholesterol. Specifically bound [³H]25-hydroxycholesterol was measured (n = 6). m, n, Top, the immunoprecipitated and purified Flag/His-tagged wild-type INSIG1 or INSIG1(S207E) (m) or Flag/His-tagged wild-type INSIG2 or INSIG2(S151E) (n) from Huh7 cells was incubated with the indicated concentration of [³H]25-hydroxycholesterol. Specifically bound [³H]25-hydroxycholesterol was measured (n = 6). **P < 0.001 (two-tailed t-test). Data in e–n are mean ± s.d. All experiments were repeated three times independently with similar results.

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