FIGURE 2.

SCF-FBXO31 ubiquitylates Cdt1. A, in vivo ubiquitylation assay was performed on HEK293T cells transfected with constructs expressing tagged proteins as shown and incubated with 10 μm MG132 for 4 h before harvesting. Whole cell extracts (bottom panel) and 8×His-Cdt1 affinity-purified (AP) on nickel-nitrilotriacetic acid Spin Columns (Qiagen) under denaturing conditions were Western-blotted (WB) with antibodies as shown. Polyubiquitylated Cdt1 appears as a ladder in lane 3 (top blot). B, for in vitro ubiquitylation assay, SCF complexes were affinity-purified with anti-HA-conjugated agarose beads from HEK293T cells expressing HA-tagged Skp1, Cul1, and Roc1 with or without FBXO31 and incubated with GST-Cdt1, E1 (UBE1), E2 (UbcH5a and UbcH8), ubiquitylation buffer, Myc-ubiquitin, and Cdk2/cyclin A. The reaction was stopped by the addition of 1× protein-loading buffer, and samples were Western-blotted with antibodies as shown. Polyubiquitylated-Cdt1 appears as a ladder in lane 2 (top blot).