Figure 1. TRIM27 binds directly to ULK1 and this binding is regulated by nutritional starvation.

• A
  Venn diagram of FIP200 binding proteins identified in MEF following FIP200 co‐immunoprecipitation analysis under normal versus starvation conditions.
• B
  Co‐immunoprecipitation assays in HEK293T cells after co‐transfection with Flag‐TRIM27 and the indicated combinations of empty HA‐vector, HA‐tagged FIP200, ULK1, or ATG13 before immunoprecipitation with anti‐HA antibodies. Immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to Western blotting against anti‐HA or Flag.
• C–F
  WT (C), FIP200 knockout (KO) (D), ULK1/2 knockout (E) MEF cells or ATG13 knockout 4T1 cells (F) were subjected to starvation conditions before immunoprecipitation against control Immunoglobulin G (IgG) or anti‐TRIM27 before blotting against the indicated endogenous forms of TRIM27, FIP200, ULK1, ATG13, and USP7.
• G
  Co‐immunoprecipitation assays in HEK293T cells after co‐transfection with Flag‐TRIM27 and either full length ULK (HA‐ULK1 1–1,051) or the indicated various truncated forms of ULK1 designated HA‐ULK1 1–828, HA‐ULK1 1–600, and HA‐ULK1 1–500; AA, amino acids.
• H
  Co‐immunoprecipitation assays in HeLa cells stably expressing HA‐ULK1 and immunoprecipitation against IgG or HA followed by Western blotting as indicated.
• I
  HeLa cells stably expressing Flag‐TRIM27 were starved as indicated or starved for 2h before replenishing with complete medium. Anti‐Flag immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to blotting against Flag and ULK1.
• J
  Purified recombinant GST or GST‐TRIM27 was incubated with recombinant HA‐ULK1 and bound ULK1 analyzed by Western blotting.
• K
  Schematic diagram of TRIM27 binding to ULK1‐ATG13‐FIP200 complex in normal or starvation conditions.

Data information: For B–J, the experiments were performed at least three times independently with similar results.

Source data are available online for this figure.