G 25 spin column

The regressed fork, i.e., Chicken-foot (CF) substrate, and replication fork (RF) substrate were prepared using oligonucleotides listed in Supplementary Table 1 as follows: For fork restoration assays, 10 pmol of oligo B was labeled at its 5’ end using T4 polynucleotide kinase and 30 µCi γ³²P-ATP in a 20 ul reaction at 37 °C for 40 min. After heat inactivation at 65 °C for 20 min, free ATP was removed by applying the sample to a G-25 spin column (Cytiva). 10 pmol of either oligo A (for CF) or oligo D (for RF) was annealed to the labeled oligo B in 50 µl total volume with 10 mM Tris–HCl, pH 7.5 and 50 mM NaCl by heating the oligonucleotides at 95 °C for 5 min followed by slow cooling to RT. 50 pmol of Oligo C and either oligo D (for CF) or A (for RF) were annealed similarly in 10 mM Tris, pH 7.5, and 50 mM NaCl. Just prior to the fork restoration assay, the CF substrate was prepared by combining 1 pmol of radiolabeled substrate AB and 2 pmol of unlabeled substrate CD in annealing buffer (10 mM Tris–HCl pH 7.5, 10 mM MgCl₂, 50 mM NaCl) and incubating at 37 °C for 30 min followed by RT for an additional 30 min. For the replication fork, radiolabeled 1 pmol of BD substrate was annealed to 2 pmol of substrate AC in a similar fashion.

Baculovirus was generated by transfecting purified bacmid DNA into Sf9 cells using FuGENE HD (E1910, Promega Corporation, Madison, WI, USA), and subsequently used to infect suspension cultures of Sf9 cells (2 × 10⁶ cells/mL) with a multiplicity of infection of 1. Infected Sf9 cells were incubated at 27 °C for 48 h for protein expression. The Sf9 cells were suspended in buffer A (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, and 1 × protease inhibitor), sonicated at 4 °C and centrifuged at 28,980× g for 20 min. Soluble protein fractions were mixed with FLAG-M2-agarose beads (A2220, Sigma-Aldrich, St. Louis, MI, USA) pre-equilibrated with buffer A. The His-FLAG-TBK1 protein was eluted using buffer A containing 3 × FLAG peptide (200 µg/mL), concentrated using an Amicon Ultra-15 centrifugal filter unit (Merck, Billerica, MA, USA), and applied to a G-25 spin column (Cytiva) pre-equilibrated with buffer including 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 × protease inhibitor, and 1 × phosphatase inhibitor (PhosSTOP, 04906837001, Roche, Basel, Switzerland).