CHO-IR cells expressing human insulin receptor are grown to 80% confluence in Hamm’s F12 medium with 10% fetal bovine serum and without hypoxanthine. Trypsinized cells are seeded in 6-well plates at 1 × 106 cells/well in 2 ml of medium without fetal bovine serum. After 24 h, medium is replaced with 1 ml of serum-free medium containing GSK-3 inhibitor or control (final DMSO concentration <%) for 30 min at 37°C. Cells are lysed and centrifuged 15 min at 4°C/14000g. The activity ratio of GS is calculated as the GS activity in the absence of glucose-6-phosphate divided by the activity in the presence of 5 mmol/l glucose-6-phosphate, using the filter paper assay of Thomas et al.
TIR1 proteins are F-box proteins that have three different domains giving them the ability to bind to three different ligands: an SCF TIR1 ubiquitin ligase complex (using the F-box domain), auxin  (so TIR1 proteins are auxin receptors), and Aux/IAA proteins (via a degron domain). Upon binding of auxin, a TIR1 protein's degron domain has increased affinity for Aux/IAA repressor proteins, which when bound to TIR1 and its SCF complex undergo ubiquitination and subsequent degradation by a proteasome . The degradation of Aux/IAA proteins frees ARF proteins to activate or repress genes at whose promoters they are bound.