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

The two fractions were precleared by incubating them 1 h with 3 µg control mouse IgG (Santa Cruz Biotechnology) and 40 µl Protein A/G Agarose. Afterwards, immunoprecipitation was performed by incubating the lysates with 6 µg anti-Flag M2 antibodies together with 80 µl Agarose beads for 4 h at 4 °C on a rotating wheel. Beads were washed five times with 2 ml IGEPAL lysis buffer, transferred to a fresh tube and proteins were eluted at 70 °C for 10 min in LDS sample buffer (Invitrogen). Samples were separated on a 4-12 % Bis-Tris gradient-gel (NuPAGE, Invitrogen), stained with colloidal Coomassie (Invitrogen) and cut into small pieces (7 slides each lane). In-gel digestion of the proteins with trypsin and purification of the peptides was performed as described (Seibert et al., 2019) . Peptide solutions were desalted by stop and go extraction (STAGE) tips (Rappsilber et al., 2003) . The purification and mass spectrometry of two individual experiments was performed with a time lag and therefore analyzed with different instrumental settings. Dissenting setting parameters of analysis 1 and 2 are indicated by a slash. Samples were eluted from STAGE tips with acetonitrile and applied to the UHPLC system (EASY-nLC 1000, Thermo Fisher Scientific) in 0.1 % formic acid. Separation of peptides by hydrophobicity was performed with 50/18 cm in-house packed C18 columns (1.9 µm C18 beads, Dr. Maisch GmbH). Peptide elution was achieved with a binary solvent system (solvent A: 0.1% formic acid; solvent B: 80% acetonitrile, 0.1% formic acid) by increasing the relative amount of B from 10 % to 38 % in a linear gradient within 35/20 min, followed by 5/3 min up to 60 % and another 5/2 min to 95%. Re-equilibration was done within 5 min at 5 %. The samples were transferred to an in line coupled QExactive orbitrap/QExactive HF mass spectrometer (Thermo Fisher Scientific) using a nano electrospray ionization source. Full MS spectra were acquired with a data-dependent Top10/15 method that comprised a resolution of 70,000/60,000 at 200 m/z and an automatic gain control (AGC) target of 3e6 at a maximum injection time of 20 ms. The 10/15 most intense ions were further fragmented with higher-energy collisional dissociation (HCD) at a normalized collision energy of 25/27 and MS² spectra were generated at 35,000/30,000 resolution, AGC target of 5e5/1e5 and maximum injection time 120/64 ms.