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Blood, Vol. 112, Issue 5, 2062-2070, September 1, 2008
HES1 is a novel interactor of the Fanconi anemia core complex
Blood Tremblay et al.
112: 2062
Supplemental materials for: Tremblay et al
Files in this Data Supplement:
- Table S1. Mass spectrometry analysis of HES1 binding proteins (PDF, 29.9 KB)
- Figure S1. Mapping binding sites for the interactions of HES1 with FANCF and FANCG (JPG, 55.4 KB)
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(A) Mapping the HES1 binding region on FANCF. (B) Mapping the HES1 binding region on FANCG. Yeast strain AH109 was co-transformed with vectors expressing full length HES1 with FANCF or FANCG constructs as indicated and assayed for interaction as described in Materials and Methods. A positive interaction is indicated as +. FANCA construct was used as positive control. Negative controls included FANCF or FANCG constructs co-transformed with pGBKT7 empty vector.
- Figure S2. HES1 co-precipitates with FA proteins (JPG, 83.5 KB)
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(A) Co-immunoprecipitation of HES1 with FANCA and FANCG. 293T cells were transfected with HA-tagged HES1 and vectors expressing FANCA and myc-FANCG and subjected to immunoprecipitation (IP) with anti-FANCA, anti-HA (HES1), anti-Myc (FANCG) or control IgG followed by immunoblotting with antibodies as indicated. (B) Co-immunoprecipitation of HES1 with FANCC. 293T cells were transfected with HA-tagged HES1 and FANCC and subjected to immunoprecipitation (IP) with anti-FANCC, anti-HA (HES1), or control IgG followed by immunoblotting with antibodies as indicated. (C) Co-immunoprecipitation of HES1 with FANCE. 293T cells were transfected with HA-tagged HES1 and myc-FANCE and subjected to immunoprecipitation (IP) with anti-myc (FANCE), anti-HA (HES1), or control IgG followed by immunoblotting with antibodies as indicated. (D) Co-immunoprecipitation of HES1 with FANCF. 293T cells were transfected with HA-tagged HES1 and myc-FANCF and subjected to immunoprecipitation (IP) with anti-myc (FANCF), anti-HA (HES1), or control IgG followed by immunoblotting with antibodies as indicated. (E) Co-immunoprecipitation of HES1 with FANCL. 293T cells were transfected with HA-tagged HES1 and flag-FANCL and subjected to immunoprecipitation (IP) with anti-flag (FANCL), anti-HA (HES1), or control IgG followed by immunoblotting with antibodies as indicated. (F) Co-immunoprecipitation of HES1 with EGFP. 293T cells were transfected with HA-tagged HES1 and EGFP and subjected to immunoprecipitation (IP) with anti-EGFP, anti-HA (HES1), or control IgG followed by immunoblotting with antibodies as indicated. (G) FA core complex components co-immunoprecipitate with HES1. 293T cells were cotransfected with HA-tagged HES1 and FA coding vectors and subjected to immunoprecipitation (IP) with either anti-FANCC, anti-HES1 antibodies or control IgG. IP were analysed by SDS-PAGE and western blotted using antibodies as indicated.
- Figure S3. HES1 colocalizes with FA core complex proteins (JPG, 116 KB)
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(A) Immunofluorescence staining of endogenous FANCA and HES1 in HeLa cells treated with the γ-secretase inhibitor L685,458 (10 uM, 5 hrs) or DMSO (control). (B) Immunofluorescence staining of endogenous FANCG and HES1 in HeLa cells treated with the γ-secretase inhibitor L685,458 (10 uM, 5 hrs) or DMSO (control). (C) Immunofluorescence staining of endogenous FANCL and HES1 in HeLa cells treated with the γ-secretase inhibitor L685,458 (10 uM, 5 hrs) or DMSO (control). (D) Co-localization of endogenous FANCA and FANCL in HeLa cells treated with γ-secretase inhibitor L685,458 (10 uM, 5 hrs) or DMSO (control). (E) Immunofluorescence staining of endogenous FANCC and HES1 in HeLa cells treated with the γ-secretase inhibitor L685,458 (10 uM, 5 hrs) or DMSO (control).
- Figure S4. HES1 depletion affects cell survival and FANCA cellular localization (JPG, 55.7 KB)
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(A) HeLa cells transfected with 3 different Stealth RNAi against HES1 or with scrambled sequences were treated with 30 and 100 nM MMC and incubated 5 days before staining. (B) Localization of endogenous FANCA in HeLa cells transfected with Stealth RNAi against HES1 and/or incubated with the γ-secretase inhibitor L685,458 and treated with 120 ng/ml MMC for 16 hrs. (C) Rad51 foci formation in HeLa cells. HeLa cells depleted of HES1 using Stealth RNAi against HES1 or the γ-secretase inhibitor L685,458 were treated with 100 mM MMC for 24 hrs and subjected to immunofluorescence analysis.
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