AMPK Methods and Protocols

3. Gently wash the pellet in 2 ml of ice-cold PBS and repeat
   centrifugation.


4. Remove PBS by aspiration and immediately resuspend the cell
   pellet in ice-cold lysis buffer. Vortex briefly and flash freeze in
   liquid nitrogen.


5. Now followsteps 4– 6 as for adherent cells (Subheading3.2).

3.4 Use of RG
Mutants to Test AMP
Dependence
of Activation
Mechanism

1. Plate cells in appropriate medium containing 10% FBS.


2. Transfect cells with DNA encoding either FLAG-tagged
   AMPK-γWT (γ1orγ2) or FLAG-tagged RG mutant (γ 1
   R299G or γ2 R531G) using your preferred transfection
   reagent, as per manufacturers’ instructions.


3. After allowing sufficient time to express the recombinant pro-
   tein (typically 36–48 h), you may wish to change the medium
   to 5 mM glucose (seeNote 7) at least 16 h prior to cell
   treatment and lysis.


4. Treat replicate plates with the compound under test. As con-
   trols, also treat plates with activators acting through known
   mechanisms such as AICAR, phenformin, or berberine (which
   will activate only WT complexes) or ADaM ligands such as
   A769662 or 991 or the Ca2+ ionophore A23187 (all of
   which will all activate both WT and RG complexes).


5. Harvest and lyse the cells as in Subheadings3.2 and 3.3.

3.5 Use of S108A
and K40A/K42A Cells
to Test Dependence
on the ADaM Site

1. Plate cells in appropriate medium containing 10% FBS.


2. Transfect cells with DNAs encoding either WT AMPK-β1orβ 1
   S108A mutant or encoding WT AMPK-α1orα1-K40A/K42A
   mutant, using preferred transfection reagent as per manufac-
   turers’ instructions. We recommend a nontoxic transfection
   reagent, such as FuGENE 6.


3. Followsteps 3– 5 in Protocol3.4.

3.6 CE and LC-MS
Assays of Adenine
Nucleotides

Steps 1– 7 below relate to sample preparation (identical for both

CE, HPLC, and LC-MS),steps 7and 8 relate to CE, andstep 9

relates to LC-MS. We have not performed these analyses by HPLC

for some time, but previously published a method for this [12].

1. Perform appropriate treatment to disturb cellular energy
   charge (including controls to establish basal levels of nucleo-
   tides) as described in Subheading3.1.


2. Lyse four dishes at appropriate time intervals, on ice, as follows:
   (a) Aspirate medium and then gently but rapidly wash plates
   in 2
   2 ml of ice-cold PBS, aspirating liquid between each
   wash and after final wash.

246 Simon A. Hawley et al.