Figure 6.

Composition of the ULK1 complex is influenced by the ATG13-ULK1 interaction but not by the LIR motif of ATG13. (A) S100 extracts of atg13 KO MEFs stably expressing the indicated HA-ATG13 variants were separated by size-exclusion chromatography on a Superose 6 increase column. Fractions were analyzed by immunoblotting for the indicated proteins. Diagrams show protein levels for each fraction at a ratio of the input and normalized to the fraction containing the highest concentration of the analyzed protein. Curves for controls (KO and ATG13) are reused in figures 2 and 5. (B) Cells described in (A) were seeded onto glass cover slips one day prior to stimulation with full medium (DMEM) or starvation medium (EBSS) for 1 h. Cells were fixed, permeabilized and stained for HA (covance MMS-101P) and RB1CC1. An inverse confocal laser scanning microscope was used for imaging. Puncta and colocalization per cell quantification was done using fiji software. A minimum of 127 cells per stimulation was analyzed. Data represent mean + SEM. Statistical analysis using the Student t test, 2-sample assuming unequal variances was performed comparing EBSS to DMEM for each individual cell line. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 20 µm. (C) atg13 KO MEFs stably expressing HA-ATG13 or the indicated mutants were seeded onto glass cover slips one day prior to stimulation with starvation medium (EBSS) for 2 h. Cells were fixed, permeabilized and stained for HA (covance MMS-101P) and LC3. An inverse confocal laser scanning microscope was used for imaging. Scale bar: 20 µm.