2.2 Methods
of Harvesting Adherent
Cells for AMPK Assays
or Monitoring Thr172
Phosphorylation
- Liquid nitrogen.
- Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
4.3 mM Na 2 HPO 4 , 1.47 mM KH 2 PO4,pH 7.4 (seeNote 1). - Lysis buffer: 50 mM Tris–HCl, pH 7.2 at room temperature,
50 mM NaF, 5 mM Na pyrophosphate, 1 mM EDTA, 1 mM
EGTA, 1% (v/v) Triton X-100, 1.3 mM dithiothreitol,
1.3 mM benzamidine, 100μM phenylmethane sulphonyl fluo-
ride, 5μg/ml soybean trypsin inhibitor (seeNote 2). - Cells in 6 cm plates at 80–90% confluency.
2.3 Methods
of Harvesting
Nonadherent Cells
(e.g., T Cells) for AMPK
Assays or Monitoring
Thr172
Phosphorylation
Materials required for this protocol are identical to those listed in
Subheading2.2; additional materials are listed below:- Cells in flask at 80–90% confluency.
- Centrifuge.
2.4 Use of RG
Plasmid to Test AMP
Dependence
of Activators
- Plasmid DNAs encoding wild-type (WT) and AMP-insensitive
forms with RG mutations affecting site 3 on the AMPK-γ
subunit (seeNote 3). - Cell line expressing LKB1 (seeNote 4), plated in 6 cm plates.
- Transfection reagent.
- Direct (e.g., salicylate, A769662, 991, MK8722) or indirect
(e.g., berberine, phenformin, troglitazone) activators of
AMPK, dissolved in water or DMSO according to solubility.
2.5 Use of S108A
and K40A/K42a Cells
to Test ADaM Site
Dependence
- Plasmid DNAs encoding wild type AMPK-β1 (WT) and S108A
mutant of AMPK-β1 (S108A mutant) (seeNote 5), or AMPK-
α1 (WT) or K40A/K42A mutant of AMPK-α1 (AA mutant).
2.6 CE and LC-MS
Assays of Adenine
Nucleotides
- Cell scraper.
- Aspirator.
- Plastic pastette.
- Four sets of labeled microcentrifuge tubes.
- PBS, ice-cold (seeNote 1).
- Perchloric acid: 5% (w/v) perchloric acid, ice-cold.
- 1:1 mixture of tri-n-octylamine and 1,1,2-
trichlorotrifluoroethane. - Chilled centrifuge.
- Capillary electrophoresis (CE) instrument. We use a Beckman
Coulter P/ACE 5500 MDQ with the following buffers:
Intact Cell Assays to Determine Contributions of AMP-Binding and ADaM Sites 243