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

To test whether the regulated solubility change of SINTBAD is associated with alterations of its intracellular distribution, immunofluorescence studies were performed in U2OS cells that are ideally suited and widely used for the characterization of subcellular compartmentation. In the absence of a commercial antibody faithfully detecting the endogenous SINTBAD protein in immunofluorescence studies, we generated SINT-BAD-deficient U2OS cells by CRISPR/Cas9-mediated genome editing ( Figures S2A and S2B ) that were A B C D Figure 1 . Stress-Inducible Formation of SINTBAD-Containing Speckles (A) HeLa cells were exposed for the indicated periods to heat shock, and cells were harvested and fractionated into cytosolic (cyto.) and nuclear/insoluble (nucl./insol.) extracts. Western blot was performed to detect the dynamic relocalization of the endogenous adapter proteins SINTBAD, AZI2, and TANK. The detection of phosphorylated ERK1/2 (T202/Y204), tubulin, and histone H3 serves as a control for the treatment, cytosolic, and nuclear/insoluble fraction, respectively. The positions of molecular weight markers are indicated. (B) 293T cells were treated with 0.5 mM arsenite for the indicated times and further analyzed as in (A).