AMPK Methods and Protocols

2 Materials

2.1 Analysis of AMPK
Activation in Cells
or Mice

1. Glucose-free DMEM: glucose-free Dulbecco’s Modified
   Eagle’s Medium (DMEM), 10% (v/v) fetal bovine serum
   (FBS) (seeNote 1).


2. Complete DMEM medium: DMEM medium (containing
   25 mM glucose), 10% (v/v) FBS.


3. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.68 mM
   KCl, 10 mM Na 2 HPO 4 , 1.76 mM KH 2 PO 4 , pH 7.4. Store
   at room temperature (RT).
   4.AXIN
   /
   ,LAMTOR1
   /
   MEFs, and the control wild-type
   MEFs (seeNotes 2and 3 ).


4. Male mice (C57BL/6 J) at 4–6 weeks old. The mice are housed
   individually (seeNote 4) for at least 24 h prior to the experi-
   ment, with free access to water and standard diet (65% carbo-
   hydrate, 11% fat, 24% protein). The light-dark cycle for mice is
   from 8 a.m. to 8 p.m.


5. Lysis buffer: 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM
   EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, 2.5 mM sodium
   pyrophosphate, 1 mM β-glycerophosphate, with protease
   inhibitor cocktail. Store at 4C(seeNote 5).


6. 2SDS sample buffer: 125 mM Tris–HCl, pH 6.8, 4% (w/v)
   SDS, 20% (v/v) glycerol, 100 mM DTT, 0.02% (w/v) bromo-
   phenol blue. Store at RT.


7. 8% SDS-PAGE gel.


8. Vertical “wet” transfer apparatus (seeNote 6).


9. Blocking solution: TBS (1Tris-buffered saline): 50 mM
   Tris–HCl, PH 7.5, 150 mM NaCl, 0.01% Tween-20, 5%
   (w/v) nonfat dry milk or bovine serum albumin (BSA).


10. Antibodies: anti-phospho-AMPKα-Thr-172, anti-AMPKα,
    anti-phospho-ACC-Ser79, and anti-ACC (seeNote 7).


11. Sonicator equipped with a 5/64^00 (2 mm) stepped microtip.


12. Electrical disperser (seeNote 8).


13. Tabletop centrifuge.

2.2 Analysis
of Lysosomal
Localization of AXIN

1. Lysosome Isolation Kit (Sigma, cat. LYSISO1) (seeNote 9),
   containing 5 Extraction Buffer, 20 OptiPrep Dilution
   Buffer, OptiPrep Density Gradient Medium, 2.5 M CaCl 2
   solution, and 2.3 M sucrose solution. Store at 4C.


2. 7-mL Dounce Glass Tissue Grinder with small clearance pestle.
   The grinder should be precooled to 4C prior to use.


3. Ultracentrifuge equipped with SW60 Ti rotor. The rotor
   should be precooled to 4C prior to use.

396 Chen-Song Zhang et al.