Fig. 4.

a Immunoblot of total AMPK following immunoprecipitation using antibodies for the α₁ or α₂ subunits of AMPK from three cultures of differentiating mESCs or from three separate litters of E7.5 or E10.5 mouse embryos, or from the skeletal muscle (S), liver (L) or heart (H) of adult animals. b AMPK activity assays were performed as in Fig. 1 following immunoprecipitation of AMPKα₁ from differentiating mESCs treated with AICAR or AA, or from the heart of an adult mouse treated with AICAR. ***p<0.001 vs control; **p<0.01 vs control. c As in b, except that the activity of α₂-immunoprecipitated AMPK was assayed. d AMPK activity in differentiating neuronal precursors derived from mESCs that had been cultured without or with AICAR, AA or compound C for 90 min. ***p<0.001 vs control, compound C; **p<0.01 vs AA; *p<0.05 vs AICAR. e Real-time RT-PCR of Pax3 mRNA normalised to rRNA from undifferentiated (UD) mESCs, or differentiating neuronal precursors (D) that were untreated, or treated with compound C, AICAR, AICAR + compound C, AA or AA + compound C. ***p<0.001 vs D, compound C, AA + compound C, AICAR + compound C. f NTDs in embryos recovered on E10.5 from mothers treated or not treated with glucose or compound C on E7.5. Pregnant mice were made hyperglycaemic as in Fig. 1. Compound C (20 mg/kg) was administered at 09:00, 13:00 and 15:00 hours. **p< 0.01 vs all other treatment groups