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

Coverslips were subsequently incubated with the indicated primary antibodies, diluted in PBS containing 1 % BSA and 0.1 % Triton X-100, for 90 min at room temperature or at 4 °C overnight. After washing three times with PBS cells were incubated with the appropriate secondary Alexa488-or Cy3-conjugated antibodies (Jackson ImmunoResearch) diluted 1:3000 in 1 % BSA in PBS for 90 min in the dark. After incubation, cells were washed three times in PBS and the nuclear DNA was stained with Hoechst 33324 (Invitrogen). The samples were mounted with Mowiol mounting medium and stored at 4 °C. Analysis of the stained cells was done using an Eclipse TE2000-E microscope (Nikon) and a 63 × oil-immersion lens. For each condition >30 healthy individual cells were analyzed and pictures of one representative cell were taken with an OCRA-spark digital CMOS camera (C11440-36U, Hamamatsu). For the quantification of protein localizations and cellular phenotypes, at least 100 cells for each condition were analyzed.