Extended Data Fig. 7: AMPK associates with the multi-enzyme complex and regulates its formation.
From: De novo pyrimidine biosynthetic complexes support cancer cell proliferation and ferroptosis defence
a, Interaction between AMPK and ectopically expressed CAD. HEK293T cells were transfected with Flag-CAD or Flag-vector as a negative control. IP analysis was performed and followed by immunoblotting. b,c, HEK293T cells expressing Flag-tagged AMPKα1 (b) or AMPKα2 (c) were subjected to IP analysis, followed by immunoblotting. d, WT and AMPKα1/2−/− Hela cells expressing Flag-CAD or Flag-vector were subjected to IP analysis with anti-Flag antibody, followed by immunoblotting. e, Interaction between endogenous AMPKα and Flag-tagged truncations of CAD. f,g, In vitro pull-down analysis of the interaction between GST-tagged AMPKα1 and His-tagged proteins. GST protein was used as a negative control. h, Interaction between Flag-tagged UMPS immunoprecipitated from HEK293T cells and GST-tagged AMPKα1 protein purified from E. coli with GST protein as a negative control. GST-pull down analysis was performed, followed by immunoblotting. i, Interaction between Flag-tagged CAD immunoprecipitated from HEK293T cells and His-tagged VDAC3 protein purified from E. coli with Flag-tagged AMPKα1 as a negative control. Ni-pull down analysis was performed, followed by immunoblotting. j, HEK293T cells were cultured under normoxia or hypoxia for 8 h. The expression of indicated proteins was detected by immunoblotting. k, WT and AMPKα1/2−/− Hela cells expressing Flag-GOT1 or Flag-vector were subjected to IP analysis with anti-Flag antibody, followed by immunoblotting. l, A schematic depicting how AMPKα interacts and regulates the pyrimidinosome. (a-k) Data are verified in three replicates with similar results.
