Figure 5. FAVL sequesters FANCL protein in the cytoplasm and promotes its degradation.

(A) FAVL and FANCL can be coimmunoprecipitated. Lysates prepared from U2OS cells transfected with empty vector or Flag-tagged FANCL or FAVL were immunoprecipitated using an anti-Flag antibody (IgG1, control). FAVL and FANCL were coimmunoprecipitated with each other (similar results are shown in Supplemental Figure 5A). (B) Overexpression of FAVL enhances levels of FANCL in the cytoplasm. A549 cells were transfected with FAVL-containing plasmid or empty vector as the control. FANCL proteins were higher in the cytoplasm of cells overexpressing FAVL but lower in total cell lysates (Supplemental Figure 5B). Brg1 shows that nuclear proteins were mostly retained in the nuclear fractions we prepared. Relative fold level increase is shown for each protein. NE, nuclear extract. (C) Downregulating FAVL enhances the steady-state levels of FANCL protein. Calu-6 cells were first transfected with control RNAi oligos or FAVL RNAi oligos. Twelve hours later, both groups of cells were transfected with an equal amount of DNA mixture, containing 10 μg Flag-FANCL and 1 μg Renilla luciferase reporter (transfection efficiency control; Supplemental Figure 5C). Levels of FANCL protein were higher in cells with low levels of FAVL at time points tested compared with cells expressing a high level of FAVL. (D) Overexpression of FAVL leads to insufficient formation of the FA complex. About 1 mg nuclear extract, prepared from U2OS cells with or without ectopically expressed FAVL, was used for FANCA IP. Levels of FANCM, FANCF, and FANCL, but not FANCA, are lower in IP pellets prepared from FAVL-elevated U2OS cells.