A549). If material is active, the user must dephosphorylate the
starting material; we recommend using PP1γand then using
okadaic acid to inhibit the phosphatase in the subsequent
assays. We have found that washing the immunoprecipitate
alone to be insufficient to completely remove PP1γ.- Thr172 phosphorylation can also be monitored by performing
secondary in-solution or IP kinase assays, as in Subheading3.4
or 3.5. This is more quantitative than Western blots, but the
experiments require careful design. If the AMPK is attached to
protein G-Sepharose beads, ensure that the beads are well
washed with assay buffer to remove upstream kinases prior to
the secondary kinase assay. If the AMPK is in-solution, it must
be diluted sufficiently in assay buffer to ensure that the carry-
over of upstream kinase into the secondary assay is negligible. - Protein for dephosphorylation protection assays must be phos-
phorylated on Thr172 and thus active. Either use bacterially
expressed AMPK that has been phosphorylated by an upstream
kinase (Subheading3.3) ensuring that the upstream kinase has
subsequently been removed, or use AMPK immunoprecipi-
tated from a cell line expressing LKB1 (e.g., HEK293, COS7
cells). Before cell lysis, the user may treat the cells with an
AMPK activator such as phenformin or berberine to cause
maximal Thr172 phosphorylation and then immunoprecipi-
tate in the presence of NaF (50 mM) to prevent
dephosphorylation.
Acknowledgment
Studies in the Hardie Laboratory were supported by a Senior
Investigator Award (097726) from the Wellcome Trust and by a
Programme Grant (C37030/A15101) from Cancer Research UK.References
- Hardie DG, Schaffer BE, Brunet A (2016)
AMPK: an energy-sensing pathway with multi-
ple inputs and outputs. Trends Cell Biol 26
(3):190–201.https://doi.org/10.1016/j.tcb.
2015.10.013 - Ross FA, MacKintosh C, Hardie DG (2016)
AMP-activated protein kinase: a cellular energy
sensor that comes in 12 flavours. FEBS J 283
(16):2987–3001. https://doi.org/10.1111/
febs.13698 - Xiao B, Sanders MJ, Carmena D, Bright NJ,
Haire LF, Underwood E, Patel BR, Heath RB,
Walker PA, Hallen S, Giordanetto F, Martin
SR, Carling D, Gamblin SJ (2013) Structural
basis of AMPK regulation by small molecule
activators. Nat Commun 4:3017.https://doi.
org/10.1038/ncomms4017- Xiao B, Sanders MJ, Underwood E, Heath R,
Mayer FV, Carmena D, Jing C, Walker PA,
Eccleston JF, Haire LF, Saiu P, Howell SA,
Aasland R, Martin SR, Carling D, Gamblin SJ
(2011) Structure of mammalian AMPK and its
regulation by ADP. Nature 472
(7342):230–233. https://doi.org/10.1038/
nature09932. nature09932 [pii] - Xiao B, Heath R, Saiu P, Leiper FC, Leone P,
Jing C, Walker PA, Haire L, Eccleston JF, Davis
CT, Martin SR, Carling D, Gamblin SJ (2007)
84 Fiona A. Fyffe et al.