Extended Data Figure 2. Glucose deprivation activates AMPK by a mechanism distinct from energy stress.

a, b, d, AXIN^-/- (a), LAMTOR1^-/- (b) and CaMKK2 knockdown (d) MEFs were incubated with DMEM containing indicated concentrations of glucose for 2 h. The levels of p-AMPKα and p-ACC in the cell lysates were analyzed by immunoblotting. c, AMP:ATP and ADP:ATP ratios in AXIN^-/- and LAMTOR1^-/- MEFs, generated as in Fig. 2a, b. e, Validation of monoclonal anti-β2 antibody for use in immunofluorescence microscopy. Parental wild type HEK293 cells, or β2 KO cells, were stained with DAPI (blue, nuclei) and anti-β2 antibody (green). Representative merged images are shown, obtained using identical intensity settings. f, Glucose starvation causes translocation of AMPKβ2 to the lysosome in HEK-293 cells that is dependent on N-myristoylation. The experiment was performed in β2 KO cells as in Fig. 1c, except that the lysosomal marker LAMP1 (tagged with RFP) was co-expressed with the WT or AMPK-β2 mutants. Upper panels show merged images stained blue [4',6-diamidino-2-phenylindole (DAPI), nuclei], red (LAMP1, lysosomes) and green (AMPK-β2, detected using antibody validated in Extended Data Fig. 2e), in cells incubated with or without glucose for 20 min. Lower small panels are magnifications of the areas indicated by dashed boxes in the upper panels, showing (L to R) red and green channels and merged images. g, Experiment identical to that shown in Fig. 2c, but expressing AMPK-β1, either WT, G2A or S108A mutants, in β1 KO cells. Results are mean ± SD, n = 4. All experiments were performed at least twice.