AMPK Methods and Protocols

5. Each reconstitution reaction requires light organelles from two
   15-cm dishes of MEFs.


6. Resuspend the light organelles with 500μL of cytosolic frac-
   tions (containing AXIN) purified from four 15-cm dishes of
   MEFs or 500μL of fractionation buffer containing 1μgof
   bacterially expressed His-AXIN at 4C.


7. Incubate the cytosolic fractions or His-AXIN with light orga-
   nelles at 37C in a thermomixer at 600 rpm for 25 min. The
   mixture can be used to determine:
   
   
   • Localization of AXIN in DRM: treat the mixture with
     1.5 mL of ice-cold Buffer A and leave the tubes on a rotator
     at 40 rpm for 1 h at 4C, and then prepare and analyze
     proteins remaining with DRM fractions by followingsteps
     21 – 23 of Subheading3.2.
   
   
   • Immunoprecipitation: lyse the mixture with 500 μLof
     ODG buffer at 4 C, and then analyze the interaction
     between LAMTOR1 and AXIN as described in steps
     33 – 36 of Subheading3.2.
   
   
   
   

Light Organelle-Based AMPK Phosphorylation Assay

The light organelles from the cell in which the lysosomal path-

way is “turned on” allows for phosphorylation of AMPK by LKB1

tethered onto the lysosomal surface by AXIN.

8. For each reaction, light organelles (one 10-cm dish of cells) are
   resuspended in 100μL of ice-cold fractionation buffer contain-
   ing 0.5μg of PP2Ac.


9. Incubate the mixtures in a thermomixer at 600 rpm for 30 min
   (seeNote 36)at32C to dephosphorylate AMPK.


10. Terminate the reaction by adding okadaic acid (10 ng/mL final
    concentration) into the mixture.


11. Add additive of interest, e.g., various concentrations of AMP
    (e.g., 0–200μM final concentrations), to the mixture contain-
    ing ATP (e.g., 200μM final concentration), and incubate the
    mixture at 30C for 20 min. The reaction is terminated by
    addition of 2 SDS sample buffer for immunoblotting
    (described insteps 7– 11 of Subheading3.1). Of note, ATP is
    included to better reflect cellular situations of the ratio of AMP
    over ATP [11].
    Light Organelle-Based ACC Phosphorylation Assay
    As described above, ACC phosphorylation (Ser-79 for ACC1
    and Ser-212 for ACC2) is primarily catalyzed by AMPK [11]. Light
    organelles purified from various treated cells are applicable for
    determining AMPK activities toward ACC in vitro.

404 Chen-Song Zhang et al.