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. 2007 Mar 29;26(8):2104–2114. doi: 10.1038/sj.emboj.7601666

Figure 2.

Figure 2

FAAP100 interacts with the FANCB and FANCL. (A) Mammalian two-hybrid assay shows direct interaction between FAAP100 and FANCB. BD-FAAP100 was cotransfected with AD-FA proteins and a luciferase reporter construct to test for direct interactions in HEK 293 cells. Fold induction of luciferase expression is relative to FAAP100 alone. Each experimental data set was obtained in triplicate. (B) Mammalian three-hybrid assay shows that the interaction between FAAP100 and FANCL depends on the presence of FANCB. HEK 293 cells stably expressing FLAG-FANCB were cotransfected with the indicated protein pairs and assayed for luciferase activation. Fold induction relative to reporter gene activation when full-length FAAP100 is expressed alone. Each experimental data set was obtained in triplicate. (C, D) Silver-stained SDS gels showing HF-FANCL-associated polypeptides purified from either cytosol (C), or nuclear extract (D), of HeLa cells by tandem-affinity purification. The major polypeptides identified by mass spectrometry are indicated by arrows. The ‘MOCK' lanes have control samples that were obtained by purification using HeLa cells that do not express HF-FANCL. The salt concentration of the washing buffer (250 or 500 mM) used in affinity purification was indicated.