Microscale thermophoresis

MST was performed as in²². Briefly, purified recombinant CSL (LabForce AG, Switzerland, TP602744), UPF1 (OriGene, USA, TP308018) and Ku70 (Origene, USA, TP304048) proteins were labelled with a RED-NHS protein labelling kit (NanoTemper). Labelled CSL protein was incubated at a constant concentration (1 µM) with two-fold serial dilutions of unlabeled UPF1 or Ku70 (from 9 µM to 0.27 nM) in standard MST buffer. Labeled UPF1 protein was incubated at a constant concentration (1 µM) with two-fold serial dilutions of unlabeled Ku70 (from 4 µM to 0.12 nM) in standard MST buffer. Equal volumes of proteins were mixed by pipetting and incubated at room temperature for 20 min. Telomere repeat duplex DNA (TTAGGG)₃ from Integrated DNA Technologies, labeled with 647-NHS, was incubated at a constant concentration (50 nM) with two-fold serial dilutions of unlabeled CSL (from 2 µM to 0.12 nM), TRF2 (from 700 to 0.02 nM), UPF1 (from 2 µM to 0.12 nM), and Ku70 (from 2 µM to 0.98 nM) in standard MST buffer. CSL specific motif duplex DNA GTTACTGTGGGAAAGAAAG and scrambled GCTACTCATACCTAGAACG (Microsynth AG) were incubated at two-fold serial dilutions (from 750 to 0.09 nM) with a constant concentration of labeled CSL (200 nM) in standard MST buffer. Mixtures were loaded in premium-treated glass capillaries (Monolith NT.115, MO-K025, NanoTemper) and loaded into the instrument (Monolith NT.115, NanoTemper). Measurement protocol times were as follows: fluorescence before 5 s, MST on 30 s, fluorescence after 5 s, and delay 25 s. Analysis was performed at 40% light-emitting diode power and 60% laser power. The K_d values were determined with the NanoTemper analysis tool.