Figure 6. Cancer-associated FANCJ CIP box mutations show reduced AND-1-binding in cells.

(A) Cancer-relevant FANCJ CIP box mutants. Amino-acid changes corresponding to FANCJ missense variants found in the indicated tumors are shown in the upper part. A multiple sequence alignment of the CIP box from various FANCJ orthologs is reported in the lower part; highly conserved amino-acid residues are highlighted in green. The following abbreviations were used: HUMAN Homo sapiens, MOUSE Mus musculus, BOVIN Bos taurus, FROG Xenopus laevis, FISH Danio rerio. (B) Co-immunoprecipitation experiments with anti-Flag agarose beads were carried out on whole extracts of HeLa FJ-KO cells transiently transfected with pCSII-EF-MCS vector expressing Flag-tagged FANCJ wild type (WT) or the indicated mutants (EV stands for empty vector). Western blot analysis was carried out on the input (1% of each sample; 5 μg of protein) and pulled-down material (20 and 40% of each sample for FANCJ-Flag and AND-1 detection, respectively). Proteins of interest were detected using the indicated antibodies. Experiments were done in triplicate. The level of immunoprecipitated endogenous AND-1 was quantitated in each sample, as described in “Methods”. Mean ± SD are shown in the bar graph, asterisk on A745T bar refers to a standard deviation of 58.44. Source data are available online for this figure.