Figure 6.

Mutant TIM proteins affect phosphorylation of TIM and PER. A, Mutant TIMs affect phosphorylation of sites recognized by PP2A. S2 cells were transiently transfected with wild-type tim or mutant tim constructs. Transfected cells were treated with PP2A inhibitor OA (500 nm), with or without proteasome inhibitor MG132 (10 μm) for 4 h. The ACTIN band is shown as a loading control. Similar results were obtained in three independent experiments. B, Effects of PP1 inhibition on phosphorylation of wild-type and mutant TIMs. S2 cells were transiently transfected with wild-type tim or mutant tim constructs. Transfected cells were treated with PP1 inhibitor TTM (4 mm), with or without proteasome inhibitor MG132 (10 μm). The ACTIN band is shown as a loading control. C, TIM mutants affect PER phosphorylation by GSK-3β. S2 cells were transfected with both per and wild-type tim (wt) or mutant tim (TA). Transfected cells were treated with varying concentrations of OA with proteasome inhibitor MG132 (10 μm), in some cases along with the GSK-3β inhibitor AR (10 μm) as indicated.