Supplementary Figure 2: Mapping the interacting regions of IFFO1 with XRCC4 and Lamin A/C.

a, Immunoblotting showing that the Superose 6 fractionation profile of IFFO1 overlaps with those of XRCC4. The fractions corresponding to molecular weight standards are indicated at the top. Inputs of some proteins were cut from the right side of the same gel. b, d Schematic representations of the different IFFO1 (b) and XRCC4 (d) deletion mutants (left) and their ability to coimmunoprecipitate with XRCC4 and IFFO1 (right), respectively. FL, full length. (c, e) Immunoprecipitation to assess whether the various deletion mutants of IFFO1 (c) and XRCC4 (e) co-purified with XRCC4 and IFFO1, respectively. NA, not available. f, Immunoblot showing the IPs of Flag-tagged Lamin A and Lamin C. g, i, Schematic representations of the different Lamin A (g) and IFFO1 (i) deletion mutants (left) and their ability to interact with IFFO1 and Lamin A (right), respectively. FL, full length. h, j, Yeast two-hybrid to assess whether the various mutants of Lamin A (h) and IFFO1 (j) interacted with IFFO1 and Lamin A, respectively. k, Schematic representations of mutations of IFFO1 in cancers. Vertical axis presents the mutation frequency of the indicated residues. l, GST-pulldown to assess whether the recurrent mutations of IFFO1 affect its interaction with Lamin A. The 1A motif of IFFO1 and 2B motif of Lamin A were fused with GST-tag and MBP-tag, respectively, and expressed in E. coli for pulldown assays. The pulldown proteins were analysed by Coomassie Blue-stained SDS–PAGE gels. m, Immunoblotting to show the immunoprecipitates of FLAG-IFFO1 wild-type and mutants using HEK293 cells. All immunoblots, gels and yeast two-hybrid are representative of three independent experiments; unprocessed scans of immunoblots are shown in Supplementary Fig. 9.