pET30 6His-TRIB2 (and various deletion or amino acid substitution constructs, including an N-terminal FLAG-tagged TRIB2, termed His-FLAG-TRIB2) and pET30a 6His-PKAc, which encodes catalytically active cAMP-dependent protein kinase domain, have been described previously (40, 49). Full-length TRIB2 was also cloned into pOPINJ to generate a His-GST-TRIB2 encoding construct for GST pulldown assays. MCL-1 and A-1210477 were prepared as previously described (61). TRIB1 (84 to 372) was a gift from P. Mace and was purified as described previously for TRIB2 (40). Briefly, protein expression was induced in BL21(DE3) pLysS bacteria with 0.4 mM isopropyl-β-d-thiogalactopyranoside, and after overnight culture at 18°C, proteins were purified to near homogeneity using an initial affinity step (immobilized metal affinity chromatography or glutathione-Sepharose chromatography) followed by size exclusion chromatography (16/600 Superdex 200) in appropriate buffers. S473D 6His-AKT1 (DU1850, amino acids 118 to 470), either active (PDK1-phosphorylated to a specific activity of 489 U/mg) or inactive, were purchased from the Division of Signal Transduction Therapy (University of Dundee) and stored at −80°C before analysis. Site-directed mutagenesis was performed as previously described (98), using KOD Hot Start DNA Polymerase (Millipore) and appropriate mutagenic primer pairs (sourced from IDT). All plasmids were Sanger-sequenced across the entire coding regions to confirm expected codon usage.

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