Sakamototide

AICAR was from Toronto Research Chemicals. A769662 was from Selleck Chemicals. Compound 13 was obtained as previously described (18 (link)). 991 (5-{[6-chloro-5-(1-methylindol-5-yl)-1H-benzimidazol-2-yl]oxy}-2-methyl-benzoic acid; CAS no. 129739-36-2) as obtained as previously described (7 (link)). Protein G Sepharose was from GE Healthcare, and FLAG-M2 resin from Sigma-Aldrich. ECL reagent and P81 filter papers were obtained from GE Healthcare. [γ-³²P]-ATP was from Perkin-Elmer. AMARA, LKBtide, and Sakamototide substrate peptides were synthesised by GL Biochem. The COS1 cell line was obtained from American Type Culture Collection, and the C₂C₁₂ cell line was obtained from Sigma-Aldrich. Frozen tissues or extracts from AMPKα1-/α2-, AMPKβ1-/β2-, and AMPKγ3-deficient mice were obtained from Benoit Viollet and Marc Foretz (Institut National de la Santé et de la Recherche Médicale, Institut Cochin, Paris, France), Gregory Steinberg (McMaster University, Hamilton, ON, Canada), and Alexander Chibalin and Juleen Zierath (Karolinska Institutet, Stockholm, Sweden), respectively. All cell culture reagents were purchased from Thermo Fisher Scientific, and all other chemicals were from Sigma-Aldrich unless otherwise stated.

Lysates (adipocytes 10–20 μg protein, adipose tissue 100–200 μg protein) were incubated with SIK2 or SIK3 antibodies coupled to protein G-Sepharose (GE Healthcare, Little Chalfont, UK), and phosphotransferase activity towards the peptide substrates HDAC5tide or Sakamototide (200 μmol/l, GL Biochem, Shanghai, China) was measured as described previously [25 (link), 32 (link)]. One unit of activity (U) was defined as that which catalysed the transfer of 1 nmol ³²P/min to the substrate.