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 prepare cell lysates under native conditions, cells were lysed on ice for 20 min in IGEPAL buffer (20 M Tris/HCl pH 7.5, 150 mM NaCl, 1 % IGEPAL CA-630 (Sigma-Aldrich), 5 % glycerol and freshly added 10 mM NaF, 0.5 mM Na 3 VO 4 , 1 mM PMSF, 5 µg/ml leupeptin and 5 µg/ml aprotinin). The lysates were cleared by centrifugation and the supernatants were transferred into a fresh tube and either used for coimmunoprecipitation studies or mixed with sample buffer for Western blot analysis. To lyse cells under denaturing conditions, the washed cell pellets were resuspended in 1  SDS sample buffer and sonicated two times for 20 sec with a Branson sonifier to shear the genomic DNA. After boiling the samples for 5 min, the lysates were analyzed by Western blotting. For subcellular fractionation experiments, cells, grown and treated in a 6 cm dish, were lysed in 160 µl low-salt buffer (10 mM Hepes, pH 7.9, added PMSF) on ice for 10 min. NP-40 (Roche) was added to a final concentration of 0.25 %, samples were briefly vortexed and centrifuged for 10 sec at 16 000 × g. The supernatants