Fig. 8: TFIP11 modulates BLM interaction at stalled forks.

A–C HeLa cells were labeled with 10 μM EdU for 15 min before treatment with 4 mM HU for 3 h. PLA was conducted with anti-BLM and anti-biotin antibodies. Representative images of PLA foci (red) (A). Scale bar, 10 μm. Quantification of the average number of PLA foci per focus-positive cell (B). Data represent means ± SD from three independent experiments. From left, n = 168, 168, 168, 168, 168, 168, 168 cells. P values were derived from a one-way ANOVA with Tukey’s multiple comparisons test. Western blot analysis of TFIP11 expression (C). Asterisk indicates a non-specific band. D SDS-PAGE profile of purified MBP-His-tagged BLM and His-SUMO-tagged TFIP11. E Schematic representation of the competition assay. F, H BLM (24 nM) was added to biotinylated splayed-arm DNA (100 nM) coupled to magnetic streptavidin beads in 20 μl of binding buffer (20 mM Tris-HCl, PH 7.5, 120 mM NaCl, 0.1% Triton X-100, 2 mM CaCl₂, 10 mM Mg(OAc)₂, 1 mM DTT, 0.1 mg/mL BSA) at 4 °C for 30 min. Excess BLM removed by magnetic separation. Then, increasing amounts of TFIP11 (F) or its mutant (12 nM, 24 nM, 48 nM) (H) was added and reactions were incubated at 4 °C for 30 min prior to separation of the DNA-bound and supernatant fractions. G BLM (24 nM) was added to biotinylated splayed-arm DNA (100 nM) coupled to magnetic streptavidin beads in 20 μl of binding buffer at 4 °C for 30 min. Excess BLM removed by magnetic separation. Then, increasing amounts of His-SUMO (24 nM, 48 nM) or TFIP11 (24 nM, 48 nM) was added and reactions were incubated at 4 °C for 30 min prior to separation of the DNA-bound and supernatant fractions. Source data are provided as a Source Data file.