SLX4 interacts with XPF/ERCC1, MUS81/EME1, and SLX1 through the MLR, SAP, and SBD domains, respectively. (A) Schematic illustration of the domain structure of SLX4 and its interacting partners, along with N-terminally HA-tagged SLX4 mutants used in this study. The broad interaction domains indicated by a thick red line were reported before.7 SV40 nuclear localization signal (NLS) was used for SLX4 (685-1834) and SLX4 (801-1834) to target them into the nucleus. SLX4 (1-671*) indicates the predicted mutant protein, p.Leu672ValfsX1198 in the cell line RA3331. (B) Analysis of the interacting partners of SLX4 mutants. The cell extracts from RA3331/E6E7/hTERT cell lines expressing the indicated SLX4 mutants were subjected to immunoprecipitation using anti-HA antibody or a control IgG. SLX4, XPF, ERCC1, and MUS81 were identified by immunoblotting with appropriate antibodies. A total of 2% of cell lysate used for immunoprecipitation was analyzed as a control (2% Input). Immunoprecipitation of the other SLX4 mutants used in this study are shown in supplemental Figure 2. (C) Cell extracts from 293T cells stably expressing GFP-SLX1 and HA-tagged SLX4 mutants were immunoprecipitated with anti-HA antibody or control IgG. SLX4 and SLX1 were identified by immunoblotting with anti-HA and anti-GFP antibodies, respectively. Asterisks indicate cross-reacting band. A total of 1% of cell lysate used for immunoprecipitation was analyzed as a control (1% Input).
