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. Author manuscript; available in PMC: 2013 Mar 14.
Published in final edited form as: Cell. 2012 Sep 14;150(6):1196–1208. doi: 10.1016/j.cell.2012.07.032

Figure 4. Ragulator preferentially interacts with nucleotide-free RagB.

Figure 4

A) EDTA increases the interaction between endogenous Ragulator and FLAG-RagB. HEK-293T cells stably expressing Flag-RagB were lysed in the absence or presence of EDTA and cell lysates and anti-FLAG immunoprecipitates analyzed by immunoblotting for the levels of the indicated proteins.

B) FLAG-p18 co-immunoprecipitates more endogenous Rags in the presence of EDTA. HEK-293T cells stably expressing FLAG-p18 were treated and analyzed as in (A).

C) Ragulator preferentially interacts with nucleotide-free Rags. In vitro binding assay in which immobilized HA-GST-Ragulator was incubated with nucleotide-free FLAG-RagB-RagC or Rag heterodimers loaded with GTP or GDP. HA-GST precipitates were analyzed for the levels of the indicated proteins.

D) Table summarizing Rag mutants used in this study.

E) The RagBT54N mutant preferentially interacts with endogenous Ragulator. Anti-FLAG immunoprecipitates were prepared from HEK-293T cells transfected with the indicated cDNAs in expression vectors and analyzed as in (A).

F) Quantification of endogenous MP1 and p18 binding to RagBT54N-RagC and RagBRagCS75N. Each value represents the normalized mean ±SD for n=3. See also Figure S4.