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The recombinant GST-dogfish NKCC1 fusion protein (10 ??g) was phosphorylated in vitro by AMPK and [??-32P]MgATP (specific radioactivity, 1000 cpm pmol?1) as described above Cyclopamine for 1 h and precipitated with 10% (w/v) TCA for digestion with trypsin overnight at 30??C (Horman et al. 2006). Peptides were separated by reverse-phase narrow-bore HPLC in a linear acetonitrile gradient at a flow rate of 200 ??l min?1 and radioactive peaks were analysed by nanoelectrospray ionization tandem mass spectrometry in a LCQ Deca XP Plus ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA, USA). Protein concentration was estimated (Lowry et al. 1951) with bovine serum albumin as standard. Data are expressed as mean ??s.e.m. A Student's two-sided t test was used to assess the statistical significance (P < 0.05) of the data. All statistical analyses were performed using GraphPad Prism version 4.00 (GraphPad CDK assay Software, San Diego, CA, USA). Anti-phospho-Thr172-AMPK?? antibody was from Cell Signaling Technology (Beverly, MA, USA) and T4 mouse monoclonal anti-NKCC antibody was from American Research Products, Inc. (EMELCA Bioscience, Antwerp, Belgium). Anti-??1 AMPK, anti-CaMKK?? and pyruvate dehydrogenase (PDH) antibodies were donated by Prof. D. G. Hardie (Dundee University, Scotland). Recombinant GST-dogfish NKCC1 (1?C260), recombinant GST-human NCC (1?C100) and anti-phospho-Thr233, anti-phospho-Ser373-SPAK, anti-total SPAK, anti-phospho-Thr203/Thr207/Thr212-NKCC1, anti-WNK and anti-OSR1 antibodies were kindly provided by Prof. D. R. Alessi (University of Dundee, Scotland). Polyclonal antibodies were raised against phosphopeptides corresponding to the sequences surrounding Ser77 and Ser242 of human Wnt inhibitor NKCC1, CPLGPTPpSQSRFQV and CGEKLLRPpSLGEFHD, respectively with an N-terminal cysteine for coupling to keyhole limpet haemocyanin and immunization in sheep (Sugden et al. 1999). Sera were first bound to columns containing the same immobilized peptides that had been used for immunization. The phospho-specific antibodies were eluted (Sugden et al. 1999) then passed through columns containing immobilized dephosphopeptides. Phospho-specific antibodies were tested for immunoreactivity by ELISA with the immunizing antigen and for specificity by immunoblotting. Anti-sheep, anti-mouse and anti-rabbit IgG conjugated to peroxidase were purchased from Sigma (St Louis, MO), Santa Cruz Biotechnology (Santa Cruz, CA, USA) and GE Healthcare (Little Chalfont, UK), respectively. A769662 was kindly given by Dr A. Balandran (AstraZeneca, M?lndal, Sweden). STO-609 was from Calbiochem (La Jolla, CA, USA) and bumetanide was from Biomol Research Laboratories Inc. (Plymouth, PA, USA). Radioactive 86Rb+ (37.0 GBq g?1) and [??-32P]ATP (111 TBq mmol?1) were from Perkin-Elmer (Billerica, MA, USA). Recombinant bacterially expressed ??1??1??1-AMPK heterotrimers were kindly provided by Dr D.

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