Figure 2.

β-TRCP mediates degradation of FANCM. (A) A schematic for FANCM protein and alignment of amino acids corresponding to the DSGxxS sequence with FANCM orthologs and other β-TRCP substrates. (B) HeLa cells were treated with indicated siRNAs before being arrested using nocodazole. The higher migrated bands of FANCM and WEE1 indicate phosphorylated forms. (C) GST-β-TRCP proteins were overexpressed in 293T cells and pull-down assay was performed using glutathione sepharose. (D) Flag-FANCM wild type or SSAA mutant were expressed in HeLa cells that were synchronized by double thymidine block followed by release into nocodazole-containing media for indicated times. (E) GST-β-TRCP1 was coexpressed with either Flag-FANCM wild type or SSAA mutant in 293T cells and pull-down assay was performed using glutathione sepharose. (F) 293T cells were cotransfected with 6XHis-Ub plasmid and Flag-FANCM wild type or SSAA plasmids, followed by nocodazole and MG132 treatment. Cells lysates were subjected to pull-down using Ni-NTA beads and the eluates were analyzed by Western blots.