Antibody for Serine 65 Phosphorylated Ubiquitin Identifies PLK1-Mediated Phosphorylation of Mitotic Proteins and APC1

Deciphering the protein posttranslational modification (PTM) code is one of the greatest
biochemical challenges of our time. Phosphorylation and ubiquitylation are key PTMs that dictate
protein function, recognition, sub-cellular localization, stability, turnover and fate. Hence, failures in
their regulation leads to various disease. Chemical protein synthesis allows preparation of ubiquitinated
and phosphorylated proteins to study their biochemical properties in great detail. However,
monitoring these modifications in intact cells or in cell extracts mostly depends on antibodies, which
often have off-target binding. Here, we report that the most widely used antibody for ubiquitin
(Ub) phosphorylated at serine 65 (pUb) has significant off-targets that appear during mitosis. These
off-targets are connected to polo-like kinase 1 (PLK1) mediated phosphorylation of cell cycle-related
proteins and the anaphase promoting complex subunit 1 (APC1).