Figure 3. The WIPI protein interactome.

Monoclonal U2OS cell lines stably expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI2D, GFP-WIPI3, GFP-WIPI4 or GFP alone were analysed by anti-GFP immunoprecipitation followed by nano liquid chromatography (LC)-MS/MS analysis (for detailed information, see Materials and Methods). (a) GeneMANIA was used to visualize identified WIPI protein–protein interactions. Red lines represent interactions identified in this study, and grey lines represent previously reported and predicted interactions (GeneMANIA, see Materials and Methods). ATG proteins and autophagy regulators are indicated with red letters. Complete results can be found in Supplementary Data 2. Interactions identified using GFP-WIPI2B and GFP-WIPI2D were combined and are presented as WIPI2 interactions. Proteins exclusively interacting with either of the two WIPI2 isoforms can be found in Supplementary Data 2. (b) A sub-network of the WIPI protein interactome focusing on autophagy-related and autophagy-relevant proteins is shown. Red lines represent interactions identified in this study, with dotted red lines representing interactions with low peptide counts in our LC-MS/MS analysis. Grey lines represent previously reported and predicted interactions (GeneMANIA, see Materials and Methods). In addition, the reported interactions of FIP200-ATG16L, TSC1-FIP200 and AMPK-TSC2 that did not appear when using GeneMANIA are also shown in grey. Supplementary Data demonstrating WIPI1, WIPI2B, WIPI2D and WIPI4 co-immunoprecipitation with NudC (Supplementary Fig. 4) is available.