Figure 2.

Atg13 interacts with ULK C-terminus and mediates the interaction between ULKs and FIP200. (a and b) Atg13 isoforms 1 and 2, but not isoform 3, bind ULK1 and ULK2. Myc-tagged Atg13 isoforms were expressed alone (a) or with HA-ULK2 (b) in 293T cells, and the amounts of endogenous ULK1 or HA-ULK2 in myc immunoprecipitaes was analyzed by Western blotting. (c) ULK1, ULK2, and FIP200 interact with Atg13 C-terminus. Myc-tagged fragments of Atg13 were coexpressed with HA-tagged ULK1, ULK2, or FIP200 in 293T cells. The levels of HA-constructs coimmunoprecipitated with myc-tagged Atg13 fragments were analyzed on Western blots. (d) Atg13 interacts with ULK C-terminus. Myc-tagged fragments of ULK1 or ULK2 were coexpressed with HA-Atg13 in 293T cells. The amount of HA-Atg13 recovered with myc-tagged constructs was analyzed on Western blots. (e) Atg13 directly interacts with ULK1. GST alone or GST-tagged ULK1 or ULK2 fragments were incubated with either E. coli–purified Atg13 or myc-Atg13 expressed in 293T cells, and the amounts of Atg13 recovered with GST-tagged proteins were analyzed by Western blotting. (f) Atg13 interacts with FIP200 directly and mediates the interaction between FIP200 and ULKs. GST-tagged ULK1 and ULK2 C-terminal fragments purified from E. coli were incubated with myc-tagged FIP200 expressed in 293T cells in the presence or absence of Atg13 purified from E. coli. The amounts of myc-FIP200 and Atg13 recovered with GST-tagged proteins were analyzed by Western blotting. (g) Atg13 is necessary for the interaction between ULK and FIP200. Myc-tagged FIP200 was coexpressed with HA-tagged ULK1 in 293T cells transduced with either scrambled shRNA or Atg13 shRNA. The amount of myc-tagged FIP200 coimmunoprecipitated with HA-tagged ULK1 in HA immunoprecipitate was analyzed by Western blotting.