γ2 (Q9UGJ0), orγ3 (Q9UGI9) were inserted into the cloning
site using restriction sites PstI and EcoRI. Addition of HA
affinity tag at the N-terminus was made possible by incorporat-
ing the relevant sequence into the forward primer used for
PCR amplification.- Mammalian cell line (COS7 or HEK293).
- DMEMþ10% FBS: Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% (v/v) fetal bovine serum
(FBS). - Penicillin/streptomycin.
- PBS: phosphate buffer saline (pH 7.4).
- Trypsin/EDTA (pH 7.4).
- Transfection reagent: FuGENE HD (Promega;seeNote 4).
- Reduced cell lysis buffer: 50 mM Tris–HCl, pH 7.4, 150 mM
NaCl, 50 mM NaF, 1 mM NaPPi, 1 mM EDTA, 1 mM EGTA,
1 mM dithiothreitol (DTT), 1% (v/v) Triton X-100, cOm-
plete™mini protease inhibitor cocktail (Sigma-Aldrich; see
Note 5).
2.2 Purification
of Recombinant AMPK
- Glutathione agarose.
- Glutathione agarose wash buffer: 50 mM Tris–HCl, pH 7.4,
150 mM NaCl, 1 mM DTT, 10% (v/v) glycerol. - Glutathione agarose elution buffer: 50 mM Tris–HCl,pH 7.4,
150 mM NaCl, 1 mM DTT, 10% (v/v) glycerol, 20 mM
glutathione (seeNote 6).
2.3 Modification
of the Protocol
for Postharvest
Increase or Decrease
of AMPK Activity
- pcDNA3(-) human CaMKK2. cDNA for human CaMKK2
isoform 1 (Q96RR4-1) was inserted into the cloning site
using restriction sites XhoI and HindIII. Addition of FLAG
affinity tag at the C-terminus was made possible by incorporat-
ing the relevant sequence into the reverse primer used for PCR
amplification. - Nonreducing cell lysis buffer: 50 mM Tris–HCl, pH 7.4,
150 mM NaCl, 50 mM NaF, 1 mM NaPPi, 1 mM EDTA,
1 mM EGTA, 1% (v/v) Triton X-100, cOmplete mini protease
inhibitor cocktail (Sigma-Aldrich;seeNote 5). - Anti-FLAG M2 affinity agarose gel (Sigma-Aldrich;seeNote 7).
- FLAG agarose wash buffer: 50 mM Tris–HCl, pH 7.4,
150 mM NaCl, 10% (v/v) glycerol. - FLAG agarose elution buffer: 50 mM Tris–HCl, pH 7.4,
150 mM NaCl, 10% (v/v) glycerol, 1 mg/ml FLAG synthetic
peptide (seeNote 8).
162 Jonathan S. Oakhill et al.