Figure 5. Stx17^pS202 interacts with ATG13 and FIP200.

(A, B) Co-IP showing interactions of endogenous ATG13 (A) or FIP200 (B) with Stx17^pS202 in 293 T cells. (C) Co-IP analysis of interactions between FLAG tagged Stx17 ^WT, Stx17 ^S202A or Stx17 ^S202D and ATG13 in 293T cells. (D) Graph showing quantifications of interactions between ATG13 and FLAG-Stx17 variants. **, p < 0.01, (n=3) ANOVA. (E) Co-IP analysis of interactions between FLAG tagged Stx17 ^WT, Stx17 ^S202A or Stx17 ^S202D with endogenous FIP200 in 293T cells. (F) Graph showing quantifications between FIP200 and FLAG-Stx17 variants. **, p < 0.01, (n=3) ANOVA. (G) Confocal microscopy analysis of colocalization between endogenous ATG13 and Stx17^pS202 in BMMs incubated with EBSS for 1h. Scale bar 5 μm. (H,I) HC microscopy analysis and quantifications of colocalization between ATG13 and Stx17^pS202 in BMMs incubated in full media or induced for autophagy by incubation with EBSS for 1h. Blue masks, algorithm-defined cell boundaries (primary objects); yellow masks, computer-identified overlap between Stx17^pS202 and ATG13 dots. **, p < 0.01, (n=3) t-test. (J) Confocal microscopy analysis of colocalization between GFP-FIP200 and Stx17^pS202 in HeLa cells incubated with EBSS for 1h. (K, L) HC microscopy analysis and quantifications showing colocalization between GFP-FIP200 and Stx17^pS202 in HeLa cells transfected with GFP-FIP200 and incubated in full media or induced for autophagy by incubation with EBSS for 1h. Blue masks, computer-identified GFP-FIP200 positive cells (primary objects); yellow masks, computer-identified overlap between Stx17^pS202 and GFP-FIP200 dots in GFP positive cells. **, p < 0.01, (n=3) t-test. (M) Confocal microscopy analysis of colocalization between GFP-ULK1 and Stx17^pS202 in HeLa cells incubated with EBSS for 1h. (N, O) High content microscopy and quantifications showing colocalization between GFPULK1 and Stx17^pS202 in HeLa cells transfected with GFP-ULKL1 and incubated in full media or induced for autophagy by incubation with EBSS for 1h. White masks, computer-identified GFP-ULK1 positive cells (primary objects); yellow masks, computer-identified overlap between Stx17^pS202 and GFP-ULK1 dots in GFP positive cells. **, p < 0.01, (n=3) t-test. Masks; white: GFP-ULK1 positive objects cells; yellow: number of Stx17^pS202 dots also positive for GFP-ULK1 dots. (P) A model showing Stx17^pS202 as an interacting partner of mPAS complex. In HC experiments (H,I,K,L,M,O), images are details from a large database of machine-collected and computer-processed images; data are from 3 independent experiments (>500 primary object examined per well; minimum number of wells, 20). See also Figure S4.