GFP-tagged Hec1 wild type, S55A or 6A (Ser55 and five other Ser/Thr residues changed to Ala) mutant was ectopically expressed in HeLa cells and the cell lysate was used for WB analysis with the Hec1 pSer55 antibody (diluted at 1:500)

Purified 6his-Hec1 was subject to a kinase assay with recombinant Aurora B and gamma-p32-labelled ATP. The resultant phospho-Hec1 were detected by measuring radioactive signal and by WB analysis with pSer55 antibody

Untreated (–) and treated (+) HeLa whole cell extracts (30 μg) were separated by 7.5% SDS-PAGE, and the membrane was blotted with Hec1 (phospho Ser 55) antibody (GTX70017) diluted at 1:500. The HRP-conjugated anti-rabbit IgG antibody (GTX213110-01) was used to detect the primary antibody.