ULK1/2 Restricts the Formation of Inducible SINT-Speckles, Membraneless Organelles Controlling the Threshold of TBK1 Activation

Saul, Vera Vivian; Seibert, Markus; Krüger, Marcus; Jeratsch, Sylvia; Kracht, Michael; Schmitz, Michael Lienhard

As none of these experiments related the SINTBAD-containing speckles to known subcellular structures, we aimed to identify the SINTBAD interactome by mass spectrometry. Cytosolic and nuclear/insoluble fractions were prepared from 293T cells, transfected with moderate levels of FLAG-tagged SINTBAD, followed by immunoprecipitation and identification of coprecipitating proteins by mass spectrometry as schematically shown in Figure 5A . For further bioinformatic analysis we considered proteins identified in two independent biological and technical replicates and defined high-confidence interactors by three different criteria as specified in detail in Figure S8A . This analysis revealed 150 high-confidence SINT-BAD interactors of which 27% were found in the cytosol, 58% in the nuclear/insoluble fraction, and 15% in both fractions. A list of the high-confidence interactors and the complete mass spectrometry dataset is given in Table S1 . To validate the interaction between SINTBAD and some of the interactors by (D) 293T cells were transfected to express FLAG-SINTBAD together with HA-HSP70. One day later, one cell dish was treated with heat shock as shown and cells were lysed. Lysates were split into two aliquots, one being used for immunoprecipitation (IP) using an anti-HA antibody. The other aliquot was used as an input control sample to ensure correct and comparable protein expression. The samples were further analyzed by western blot using appropriate antibodies as indicated.