Figure 5.

Deletion of FANCC Does Not Impact the In Vitro Ubiquitin Ligase Activity of the FA Core Complex, and the FANCB-FANCL-FAAP100 Subcomplex Is Essential for Monoubiquitination In Vivo

(A) Western blots of ΔFANCB/FANCB-GS (ΔB/B-GS) and ΔFANCB/FANCB-GS/ΔFANCC (ΔB/B-GS/ΔC) cells (with and without MMC treatment) after subcellular fractionation into cytonucleoplasmic (S) and chromatin (P) fractions with anti-FANCD2. Low and high exposures are shown.

(B) SDS-PAGE and silver stain analysis of the FA core complex purified from ΔB/B-GS and ΔB/B-GS/ΔC cell lines in parallel. The inset shows loss of FANCC and FANCE proteins and reduction of FANCF.

(C) In vitro ubiquitination assays of the FA core complexes (∼80 nM) shown in (B) monitored by western blotting against FANCD2 and FANCI (anti-His) and ubiquitin (anti-HA).

(D) DT40 cell lines with FA core complex subunit deletions were treated with MMC, fractionated into cytonucleoplasmic (S) and chromatin (P) fractions and subjected to western blotting with anti-FANCD2. An additional high exposure is shown for the bottom panel.

(E and F) Cellular sensitivity of WT, ΔFANCA, ΔFANCG, ΔFANCB, ΔFANCC, and ΔFANCF DT40 cells after exposure to indicated doses of MMC. The mean percentage of survival ± SEM of three independent colony-formation assays are plotted relative to untreated cells. ΔFANCB cl. 31 is the unloxed parental line to ΔFANCB cl. 5.

See also Figure S4.