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. 2020 Dec;6(6):a005595. doi: 10.1101/mcs.a005595

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© 2020 Lach et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed under the terms of the Creative Commons Attribution-NonCommercial License, which permits reuse and redistribution, except for commercial purposes, provided that the original author and source are credited.

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Figure 2. — Overexpression of FANCA R1400H rescues hypersensitivity to mitomycin C (MMC), FANCD2 ubiquitination, and localization of FANCD2 to sites of damage. (A) MMC cellular sensitivity assay of proband RA3518 fibroblasts with no vector expression (parental), expressing empty vector (EV), or overexpression of FANCA mutant (FANCA^R1400H) or wild-type FANCA (FANCA^WT). (B) Western blot assessing FANCD2 monoubiquitination and level of HA tagged FANCA expression in parental (no vector) and EV, FANCA^R1400H, and FANCA^WT expressing RA3518 fibroblasts. Cells were either untreated or cultured with 1 µM MMC for 24 h. (C) Immunofluorescence images with either anti-HA to detect overexpressed FANCA or anti-FANCD2 to detect FANCD2 localization to chromatin after treatment with 1 µM MMC for 24 h in RA3518 fibroblasts expressing EV, or overexpression of wild-type FANCA (FANCA^WT) or FANCA mutant (FANCA^R1400H). (D) MMC cellular sensitivity assay of FANCA-deficient RA3087 fibroblasts either expressing EV, a FANCA mutant (FANCA^R1400H or FANCA^F1263del), or wild-type FANCA (FANCA^WT). (E) Western blot assessing FANCD2 monoubiquitination in FANCA deficient RA3087 fibroblasts overexpressing p.F1263del FANCA. Cells were either untreated or cultured with 1 µM MMC for 24 h. Relative ratio of monoubiquitinated to nonubiquitinated FANCD2 was measured for each variant. (F) Western blot assessing level of HA-tagged FANCA expression in RA3087 cells.