Fig. 1
HES1 mono-ubiquitination and its dependence on a functional FA complex. a In vivo ubiquitination of HES1. 293T and PD430T (FA-A) cells expressing HA-HES1 and Myc-tagged ubiquitin (pCW7) or the Myc-tagged ubiquitin K48R mutant (pCW8) were subjected to immunoprecipitation using anti-Myc antibodies or control IgG and were immunoblotted against HES1 and FANCD2. b Complementation of FA-A cells restores HES1 ubiquitination. PD430T (FA-A) cells and PD430T complemented with FANCA were transfected with HA-HES1 and the Myc-tagged ubiquitin K48R mutant (pCW8). Cell lysates were subjected to immunoprecipitation with anti-Myc or anti-ubiquitin antibodies. Western blotting was performed with the indicated antibodies. c Analysis of endogenous HES1 ubiquitination in FANCA mutant fibroblasts (PD430T). PD430T cells and PD430T complemented with FANCA were transfected with the Myc-tagged ubiquitin K48R mutant (pCW8) and were subjected to immunoprecipitation with anti-Myc antibodies. Western blotting was performed with the indicated antibodies. d Immunoprecipitation of HA-HES1 using anti-Myc or anti-HA antibodies. 293T cells transfected with HA-HES1 and the Myc-tagged ubiquitin K48R mutant (pCW8) were subjected to immunoprecipitation with anti-HA or anti-Myc antibodies (left panel). Immunoprecipitation of endogenous proteins using anti-ubiquitin antibodies (right panel). Western blotting was performed with the indicated antibodies. e Alignment of the conserved region of HES1, FANCD2 and FANCI containing the putative FA recognition sequence. The peptidic sequence of HES1, FANCD2 and FANCI possess a V(I/L)XK sequence highly conserved through evolution. f HES1 lysine 109 is crucial for HES1 mono-ubiquitination. 293T cells were transfected with HA-HES1 or HA-HES1K109E coding vectors with the Myc-tagged ubiquitin K48R mutant (pCW8). Cell lysates were subjected to immunoprecipitation with anti-Myc antibodies or IgG control. Western blotting was performed with anti-HA antibodies. Antibody dilutions were used as follows: anti-HA at 1:5000; anti-HES1 at 1:1000; anti-FANCD2 at 1:1000; anti-tubulin at 1:10,000; anti-Myc at 1:500; anti-ubiquitin at 1:500; followed by secondary antibodies, anti-mouse, 1:10,000; anti-rabbit, 1:20,000