Pgs s641

Typically, 10 µg of protein was resolved by SDS–PAGE, transferred to PVDF membranes and immunoblotted as previously described [5 (link)]. Antibodies detecting TRARG1 (sc-292062 or sc-377025) and 14-3-3 (sc-1657) were from Santa Cruz Biotechnology. Antibodies against pHSL S660 (#4126), pp90RSK (#9344), p4EBP1 S65 (#9456), pTBC1D4 T642 (#4288S), pAKT S473 (#4051), pAKT T308 (#9275L), AKT (#4685), HA (#C29F4), GSK3α (#9338S), GSK3β (#9832S), pGSK3 Ser 9/21 (#8566S), pGS S641 (#3891) and Caveolin1 (#3267) were purchased from Cell Signaling Technology. Anti-α-tubulin (#T9026) was from Sigma–Aldrich. Antibody against TBC1D4 was produced as previously described [4 (link)].

Total GN antibody (S197C, third bleed) was obtained from MRC-PPU Reagents and Services. Total GS (#3893) and pGS S641 (#47043) antibodies were from Cell Signaling Technologies. pGS S641/S645 (#07-817) is from MerckMillipore. Affinity-purified pGS S8 antibody (YZ5716) was custom-generated by (YenZym Antibodies Brisbane, CA, USA) by immunisation with a combination of phosphorylated peptides of the mouse GS1 (residues 2–14: PLSRSL-*S-VSSLPG-Ahx-C-amide, in which the prefix * denotes the phosphorylated residue) and human GS1 (residues 2–14: PLNRTL-*S-MSSLPG-Ahx-C-amide). Ahx and Cysteine (C) were added at the C terminal of the antigen peptides as linker/spacer and for conjugation to carrier protein, respectively. Antibody validation is shown in Supplementary Fig. 4c. Secondary antibodies (#711-035-152 and #713-035-147) were obtained from BioRad. Glucose-6-phosphate (G6P) (#10127647001) is from Roche. All other chemicals if not noted otherwise are from Sigma Aldrich.

Cellular protein was extracted from cultured cells and fresh xenograft tumor specimens using lysis buffer (CelLytic MT; Sigma-Aldrich) containing a mixture of protease and phosphatase inhibitors (Sigma-Aldrich). A 20 μg-aliquot of protein extract was analyzed by Western blotting for the proteins of interest^{29 (link)}. The amount of protein in each sample was monitored by expression of β-actin. The following primary antibodies were used at the dilutions shown against both GSK3 isoforms (GSK3α and GSK3β; 1:1,000; Millipore), GSK3β (1:1,000; BD Biosciences) and GSK3β fractions that are phosphorylated at the serine (S) 9 residue (pGSK3β^S9; 1:1,000; Cell Signaling Technology) and the tyrosine (Y) 216 residue (pGSK3β^Y216; 1:1,000; BD Biosciences); glycogen synthase (GS; 1:1,000; Cell Signaling Technology) and its fraction phosphorylated at the S641 residue (pGS^S641; 1:1,000; Cell Signaling Technology); cyclin D1 (1:1,000; MBL); cyclin-dependent kinase (CDK)4 (1:1,000; Abcam), cyclin B1 (1:1,000, Cell Signaling Technology), poly[ADP]-ribose polymerase (PARP; 1:1,000; Cell Signaling Technology), cleaved (c)-PARP (1:1,000; Cell Signaling Technology), and β-actin (1:4,000; Ambion).