Distinct OGT-Binding Sites Promote HCF-1 Cleavage
Fig 3
The Region II CEE represents an OGT-binding sequence.
(A) Region II enhances OGT–HCF-1rep1 binding. Full-length (FL) and deletion HCF-1rep1 constructs were tested for OGT binding in the presence of UDP-GlcNAc using an in vitro OGT-directed pull-down assay. Detection of OGT and HCF-1rep1 was performed, using the indicated antibodies. Shown are 100% of OGT pull-down (panels a and b) and 11% of the input (panels c and d). *, IgG heavy chain. (B) HCF-1PRO-repeat-independent OGT–Region II binding. (Left) Schematics of the HCF-1 constructs used in this experiment. (Right) HCF-1rep1 containing Region II and an OGT-binding defective HCF-1PRO repeat (+II_T17–22A), or GST-fusion constructs containing Region II (wild-type or scrambled) alone or Region III alone (II_alone, II_scramb_alone, III_alone) were tested for binding with wild-type (WT) (left panel) or 5N-5A mutant (right panel) OGT. HCF-1 binding was detected as in (A). In (A) and (B), weak (⭕) and effective (●) OGT binding is indicated.