Figure 1
Interaction between FANCD2 and ATP5α. (A) Simple blue-staining of an 8–16% gradient gel shows “an additional extra band” in the “wtFANCD2”-lane but not in the other lanes. Flag-IP elutes were resolved in an 8–16% gradient gel, which were prepared from Flag-vector, Flag-wtFANCD2 or mtFANCD2-containing plasmids transfected 293T cells. “The extra band” pointed by a red arrowhead was sliced for mass spectrometry. (B) Confirmation of the interaction between monoubiquitinated FANCD2 and ATP5α. 293T cells were transiently co-transfected with GFP-fused ATP5α and Flag-wtFANCD2 or Flag-mtFANCD2 K561R. Cell lysate was prepared 48 hr post-transfection. Both Flag and GFP antibodies were used to perform reverse immunoprecipitation (IP) and Western blotting (WB). As red arrowheads indicated, Flag-mtFANCD2, unlike Flag-wtFANCD2, is not clearly associated with GFP-ATP5α (The equal transfection efficiency of 293T cells with GFP-fused ATP5α and Flag-wt or mt FANCD2 is shown in Supplementary Fig. 1A and B). (C) wtFANCD2 and ATP5α proteins peak at the same gel-filtration fractions prepared from control cells but not cells carrying silenced FANCL, which leads to a compromised FA-complex E3 and compromised monoubiquitination/activation of FANCD2. The sepharose 6B chromatography (gel filtration) was performed using U2OS cells stably transfected with empty vector or Lentivirus-FANCL-shRNA (Supplementary Fig. 1C). The peak of endogenous FANCD2 overlapped with the one of endogenous ATP5α protein in cells carrying a normal basal level of FANCD2 monoubiquitination, but not in the cells carrying a compromised basal level of FANCD2 monoubiquitination (resulting from silenced FANCL) (Fractions #15 and #39 correspond to the size markers of 2000 kDa and 669 kDa respectively).
