IQGAP1 interacts with Borealin both in vivo
and in vitro.
(a). Tandem affinity purification of Borealin-containing protein complexes was conducted using HeLa cells stably expressing Flag-HA- (FH-) Borealin and the control group with an empty vector. The protein complex was separated by SDS-PAGE and visualized by Coomassie Blue (CB) staining. The proteins and the number of peptides identified by mass spectrometry are shown in Table 1
. (b) Purified His-Borealin from bacteria was incubated with GST-IQGAP or GST that was prebound to glutathione agarose beads. Half of the binding proteins from these beads were separated by SDS-PAGE and visualized by CB, and half were detected by western blotting with anti-His antibody. (c) Purified His-Borealin was separated by SDS-PAGE and visualized by CB. (d) The endogenous Borealin was immunoprecipitated by anti-Borealin antibody, and the immunoprecipitates were detected by western blotting with the indicated antibodies. Endogenous IQGAP1 was immunoprecipitated by anti-IQGAP1 antibody, and the immunoprecipitates were detected by western blotting with the indicated antibodies. Three independent biological replicates were performed in the purification of recombinant protein and immunoprecipitation experiments.