Figure 6

Figure 6 Ligase activity, interaction, and subcellular localization studies with FAAP100^T542P.

(A) Reconstitution of FANCD2 monoubiquitylation using purified proteins, including HA-ubiquitin, UBE1, UBE2T, and FANCD2-FANCI complex and, as indicated, FAAP100^WT or FAAP100^T542P, FANCB, and/or FANCL. FANCD2 immunoblot: ◄D2-Ub, monoubiquitylated; ◄D2, nonubiquitylated. (B) Quantitation of results from A. FANCD2-Ub divided by total FANCD2 (percentage) indicates the ubiquitylation efficiency. Lane numbers are identical to those in A. Box plots: single value (♦), median (─), mean (□), IQR (─), minimum (x), and maximum (x) from 3 independent experiments. ***P < 0.001, by 1-way, repeated-measures ANOVA with post hoc Tukey’s test. (C) Titration of FAAP100^WT (blue) and FAAP100^T542P (red). For reaction mixtures, see A, including FAAP100^WT or FAAP100^T542P, FANCB, and FANCL. (D) Quantitation of results from C (see B and C for details). Data indicate the mean ± SD of 3 independent experiments. (E) In mammalian 2-hybrid assays, FAAP100^WT, but not FAAP100^T542P, interacted with FANCB. FAAP100 fused to the activation domain (orange) or the DNA-binding domain (red). Neither direction of FAAP100 fusion directly interacted with FANCL. Box plots: single value (♦), median (─), mean (□), IQR (─), minimum (x), and maximum (x) of 3 independent experiments. Induction factor, multiples of negative control. *P < 0.05 and **P < 0.01, by 1-way, repeated-measures ANOVA with post hoc Tukey’s test. (F) In mammalian 3-hybrid assays, FAAP100^WT, but not FAAP100^T542P, interacts with FANCL in both fusion directions in the presence of stably overexpressed FANCB. Controls, calculations, and statistical tests are the same as in E. *P < 0.05 and **P < 0.01, by 1-way, repeated-measures ANOVA with post hoc Tukey’s test. (G and H) Co-IPs from cells transfected with FANCB vector and exposed to MMC (40 ng/mL,16 hours). FAAP100^T542P in 1176 cells and FAAP100^L543_S551del in HEK293T clone Cr12 did not interact with FANCB. Transduced FAAP100^WT rescued the pull-down of FAAP100 by FANCB. Vinculin was used as a loading control. (I) On subcellular protein fractionation, FAAP100^T542P in 1176 fibroblasts and FAAP100^L543_S551del in the HEK293T clone Cr12 were not detected in nuclear extracts (NE) or on chromatin (CB). Transduced FAAP100^WT rescuedFAAP100 relocalization. CE, cytoplasmic extracts. Tubulin, YY1, and histone H3 were used as loading controls. Cells were exposed to MMC (40 ng/mL, 16 hours).