Figure 6. Copper-deficient SCO1 is unable to form SCO1-LKB1-AMPK complex and dampens AMPK activation.

(A) Immunoblotting for LKB1, AMPKα, and Flag-tagged SCO1 proteins after immunoprecipitation of SCO1 from HEK293T cells overexpressing Cp and Flag-tagged SCO1.

(B) Immunoblotting for endogenous SCO1, AMPKα, and LKB1 after immunoprecipitation of SCO1 from L02 cells treated with BCS for 24 h.

(C) Immunoblotting in non-reducing SDS-PAGE for the protein levels of copper-loaded SCO1 (Cu-SCO1) and copper-deficient SCO1 (apo-SCO1) in HEK293T cells transfected with WT or mutant (C152/156A) SCO1, followed by treatment with CuSO₄ (50 mM) or BCS (200 mM) for 24 h.

(D) Immunoblotting for AMPKα, LKB1, and Flag-tagged SCO1 proteins after immunoprecipitation of SCO1 from HEK293T cells transfected with indicated vectors; arrow indicates SCO1.

(E) Direct interaction between AMPKα and full-length SCO1 (His-SCO1) or truncated SCO1 (His-Truncated). Pull-down assays were performed with Ni-NTA beads against SCO1, followed by immunoblotting with the antibodies indicated.

(F and G) Immunoprecipitation of FLAG M2 beads against SCO1 (F) or pull-down assays with Ni-NTA beads against SCO1 (G) were performed between LKB1 and full-length SCO1 or truncated SCO1, followed by immunoblotting with the antibodies indicated.

(H) Immunoblotting for p-AMPKα, Sco1, and Cp in primary hepatocytes treated with siRNA targeting Cp combined with overexpression of full-length SCO1 (SCO1-FL) or truncated SCO1 (SCO1-Truncated).

(I) Pull-down assays were performed between AMPKα with truncated SCO1 (His-Truncated) after depleting copper binding to His-truncated with EDTA, or loading copper to His-Truncated with CuSO₄. D-isoascorbic acid (DIA) was used to reduce Cu²⁺ to Cu¹⁺. Immunoblotting in non-reducing SDS-PAGE for the protein levels of copper-loaded His-Truncated (Cu-Truncated) and copper-deficient His-Truncated (apo-Truncated).