Figure 6.

ULK1 causes loss of Raptor substrate binding without affecting mTORC1 association. (A) HA-Raptor was expressed in the presence or absence of V5-ULK1. Following HA-immunoprecipitation, western blotting was used to probe for the amount of endogenous mTOR and mLST8 co-immunoprecipitating with Raptor. (B) Raptor substrate binding was assessed using a Raptor overlay assay. 750 ng GST-4E-BP1 was resolved by SDS-PAGE and transferred to PVDF membrane. This was incubated with Myc-Raptor-containing lysate and the amount of Myc-Raptor bound to 4E-BP1 determined by western blotting with Myc antibodies. The graph shows the combined densitometry data of Raptor-4E-BP1 binding for three experiments (mean ± SD). Statistical significance was analyzed using a student's t-test, *p < 0.01. Anti-GST and anti-V5 antibodies were used to obtain total levels of GST-4E-BP1 and V5-ULK1 respectively.