For immunoblotting, cells were lysed in Laemmli buffer (10% glycerol, 1.2% SDS, 0.02% bromophenol blue, 2% β-mercaptoethanol) and boiled for 5 min. For mitotic cell extracts, cell lines were treated overnight with 200 ng/ml nocodazole (Sigma M1404) before lysis. Samples were then subjected to SDS-PAGE in a 4–20% polyacrylamide gradient gel (Biorad) and transferred to polyvinylidene difluoride (PVDF) membranes (Biorad). Membranes were incubated overnight at 4 °C with primary antibodies and then for 1 h at room temperature with HRP-conjugated secondary antibodies, according to standard procedures.
Phosphorylated forms of CENP-A from CENP-A-HA-WT and CENP-HA-AS7A cell lines were separated on a 16 cm 20% polyacrylamide (29:1 acrylamide/bisacrylamide) gel. Electrophoresis was carried out in Tris-glycine buffer for 16 h at 4 °C. The resulting bands were transferred to PVDF membranes (Bio-Rad) and immunoblotted with anti-HA antibodies. WB signal detection was performed on a Luminescent Image Analyzer–Image Quant Las 4000 mini (GE HealthCare), according to the manufacturer’s instructions. Results were obtained three times in triplicate. Uncropped gel scans are shown in the Supplementary Information (Supplementary Fig.9 and 10 ).
Phosphorylated forms of CENP-A from CENP-A-HA-WT and CENP-HA-AS7A cell lines were separated on a 16 cm 20% polyacrylamide (29:1 acrylamide/bisacrylamide) gel. Electrophoresis was carried out in Tris-glycine buffer for 16 h at 4 °C. The resulting bands were transferred to PVDF membranes (Bio-Rad) and immunoblotted with anti-HA antibodies. WB signal detection was performed on a Luminescent Image Analyzer–Image Quant Las 4000 mini (GE HealthCare), according to the manufacturer’s instructions. Results were obtained three times in triplicate. Uncropped gel scans are shown in the Supplementary Information (Supplementary Fig.