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. 2015 Apr 10;34(10):1385–1398. doi: 10.15252/embj.201490376

Figure 4.

Figure 4

Mapping the UBL5-interacting region of FANCI
  • A Diagram of FANCI fragments used to test interaction with UBL5. The UBL5-binding region is highlighted in red.
  • B Whole-cell extracts (WCE) of HeLa cells co-transfected with Strep-HA-UBL5 and the indicated GFP-FANCI fragments were subjected to GFP immunoprecipitation followed by immunoblotting with HA and GFP antibodies.
  • C, D As in (B), using the indicated GFP-FANCI constructs.
  • E Sequence alignment of amino acids in the UBL5-interacting region in FANCI proteins from different organisms. Amino acid substitutions of the highly conserved E268 and H274 residues are indicated in red.
  • F Whole-cell extracts (WCE) of HeLa cells co-transfected with Strep-HA-UBL5 and the indicated GFP-tagged FANCI constructs were subjected to GFP immunoprecipitation followed by immunoblotting with HA and GFP antibodies.
  • G Strep-GFP-FANCI WT or ∆UBL5 purified from HEK293T cells under stringent conditions was used to pull down recombinant His6-UBL5. Input and pull-down samples were subjected to immunoblotting with His6 and GFP antibodies. * non-specific band.