(A) Kymographs of Cy5-labeled IFI16 (magenta) binding to λdsDNA with varying numbers of nucleosomes tagged with anti-H4-Atto488 (blue). The number of nucleosomes per λdsDNA were estimated by quantifying IFI16 clustering sites for the lowest nucleosome/λdsDNA ratio (Figure 4—figure supplement 3C), yielding ~2 nucleosomes/ λdsDNA. At low nucleosome concentrations (~2 to 6 nucleosomes/DNA), IFI16 binds to λdsDNA and diffuses with the flow direction, until encountering a nucleosome. At higher concentrations (~20 nucleosomes per λdsDNA), individual IFI16 show only very short diffusive movements upon binding. (B) On naked λdsDNA, IFI16 travels with the flow to the free tip (top), whereas it oligomerizes along the path on nucleosome-loaded λdsDNA (bottom). (C) The overall travel distance of single IFI16 on nucleosome-loaded λdsDNA is reduced compared to bare λdsDNA (nucleosomal λdsDNA: N = 167, bare λdsDNA: N = 141).