Figure 5. SCO1 tethers LKB1 to AMPK and activates AMPK.
(A) Immunofluorescence of SCO1 and β-actin. Cells were reacted with indicated antibodies. Nuclei were counterstained with DAPI.
(B) Immunoblotting for LKB1, AMPKα, and Flag-tagged SCO1 proteins after immunoprecipitation of SCO1 from HEK293T cells. Triangle indicates LKB1 (up) and SCO1 (down).
(C) Immunoblotting for SCO1, LKB1, and AMPKα proteins after immunoprecipitation of AMPKα1 from HEK293T cells.
(D) AMPKα and SCO1 protein levels after immunoprecipitation of SCO1 from L02 cells.
(E) Immunoblotting for AMPKα, AMPKβ1/2, AMPKγ2, LKB1, and Flag-tagged SCO1 proteins after immunoprecipitation of SCO1 from HEK293T cells.
(F–I) Direct interaction between SCO1 and AMPK complex. Pull-down assays were performed with Ni-NTA beads against His-SCO1 (F, G) or an anti-Flag antibody against Flag-tagged AMPKα (H, I), followed by immunoblotting with the antibodies as indicated.
(J) Immunofluorescence of Sco1 and AMPK. Cells were reacted with indicated antibodies. Nuclei were counterstained with DAPI.
(K) Immunoblotting for p-AMPKα, ACC1, and LKB1 in primary hepatocytes treated with siRNA targeting Lkb1 (siLkb1#1, siLkb1#2) combined with CuSO4 (320 nM) treatment for 4 h.
(L–N) Immunoblotting for endogenous Sco1 and AMPKα after immunoprecipitation of Sco1 in different subcellular fraction.
(O) Protein levels of Lkb1, AMPKα, and Sco1 after immunoprecipitation of Sco1 from primary hepatocytes isolated from AMPKαf/f and AMPKα-DKO mice.