Fig. 2: Bora promotes PLK1 dimerization in vitro and in vivo.

A Bora promotes PLK1 dimerization. (Upper) HEK293T cells were transfected with increasing amounts of Bora siRNA and 24 h later transfected with Myc- and V5-PLK1. (Lower) Lysates were subjected to co-IP using V5- antibodies and western blotting using Bora-, V5, Myc- or ß-Actin-antibodies. B Size exclusion chromatography (SEC) of endogenous Bora. PLK1–3xMyc cells were synchronized to the G1/S boundary using thymidine and released for 3 h or 9 h or synchronized into the G2-phase using the CDK1 inhibitor RO3306. (Upper left panels) Lysates of the synchronized cells were subjected to an SDS-PAGE and stained with anti-PLK1, and anti-Bora antibodies. (Lower left panels) The lysates were subjected to IP using Myc-specific antibodies followed by western blotting with anti-Bora or PLK1 antibodies. (Upper right panels) The protein extracts of synchronized PLK1–3xMyc cells were loaded onto a Superose 3.2/300 size exclusion column and fractionated at a flow rate of 50 µl/min. The factions were resolved using an SDS-PAGE and probed with anti-Bora antibodies. (Lower right panel) The cell cycle distribution of synchronized HeLa cells is represented as a bar graph. (n = 3).