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. 2012 May 29;444(Pt 3):503–514. doi: 10.1042/BJ20111932

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© 2012 The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Licence (CC-BY)(http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

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(A) Purified wild-type or kinase-inactive (Lys49Met, not shown) GST–SIK2 (10 ng) expressed in HEK-293 cells was assayed in vitro using the following substrate concentrations: 0, 10, 25, 50, 100, 200 and 500 μM. (B and C) Endogenous SIK2 or AMPKα1 immunoprecipitated from 3T3-L1 adipocytes was used to evaluate the peptide substrates under the same conditions as for GST–SIK2. Results are means±S.D. for a triplicate assay. (D) Non-linear regression using a Michaelis–Menten model was used for determining V_max and K_m values using GraphPad Prism 5.