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. Author manuscript; available in PMC: 2015 Nov 20.
Published in final edited form as: Cell Rep. 2014 Nov 20;9(4):1281–1291. doi: 10.1016/j.celrep.2014.10.019

Figure 2. Sesn2 Does not Disrupt GATOR1–GATOR2 Interactions and Does not Interact with the Separate GATOR2 Components.

Figure 2

(A) Sesn2 interacts with GATOR1 in the presence of GATOR2. HEK293T cells were co-transfected with either Flag-Sesn2- or GFP-expressing constructs with all HA-tagged GATOR-expressing plasmids. Flag-Sesn2 was immunoprecipitated with anti-Flag beads and the proteins within Sesn2 complex and in the cell lysates were analyzed by immunoblotting with anti-Flag or anti-HA antibodies. (B) Overexpression of Sesn2 does not affect GATOR1–GATOR2 interactions. All GATOR proteins were ectopically expressed in the presence of either Sesn2 or GFP, immunoprecipitated via Flag-Nprl2 with anti-Flag beads and analyzed by immunoblotting as in (A). (C) Sesn2 does not affect interaction between GATOR1 and GATOR2 in vitro. All GATOR proteins were ectopically expressed in HEK293T cells. Immunocomplexes containing Flag-Nprl2 and other HA-tagged GATOR proteins were isolated with anti-Flag beads, incubated overnight with bacterially purified GST-Sesn2 and examined by immunoblotting as in (A). (D) Overexpression of Sesn2 does not have a strong impact on the GATOR-RagA:B interactions. HEK293T cells were infected with either Sesn2- or control GFP-expressing constructs and Rag complexes were immunoprecipitated with anti-RagA or control anti-PDGFRβ antibody and analyzed by immunoblotting with anti-RagA and anti-Mios antibodies. (E) Sesn2 interacts with whole GATOR2 but not its separate components. Flag-Sesn2 expressing construct was co-transfected with different combinations of the components of GATOR2-expressing constructs (HA-WDR59, HA-WDR24, HA-Mios, HA-Seh1L and HA-Sec13) into HEK293T cells. Flag-Sesn2 complexes were pulled down with anti-Flag beads and analyzed by immunoblotting as in (A). (F) Sesn2 interacts with a combination of WDR24 and Seh1L proteins. Flag-Sesn2 expressing plasmid was co-transfected with different combinations of WDR24-, Seh1L- and WDR59-expressing constructs. The Flag-Sesn2 complexes were immunoprecipitated with anti-Flag beads and analyzed by immunoblotting as in (A).