Full-length and mutant CAR sequences were cloned in-frame into pHR9SIN-SEW lentiviral expression vector, which was a gift from A. Thrasher (Institute of Child Health, University College London), and into pGEX-2T. Phospho-mutant CAR constructs were generated using site-directed mutagenesis and have been described previously (7). Control and CAR knockdown shRNA vectors (shA and shB) were in pLKO.1 backbones purchased from Sigma MISSION collection (clone ID NM_001338). H2BK-GFP and mCherry plasmids and β-tubulin–GFP were gifts from J. Monypenny (King’s College London). FLAG-tagged KIF22 constructs were a gift from M. Subba Reddy [Centre for DNA Fingerprinting and Diagnostics (CDFD) (27)]. The complementary DNAs (cDNAs) encoding the GST-NT- KIF22 and GST-CT-KIF22 domains were provided by A. Wilde (University of Toronto). The CCD of human KIF22 was cloned in pHTC HaloTag CMV-neo Vector (a gift from M. Dodding, King’s College London) between Nhe I and Xho I sites. The following primers were used for polymerase chain reaction: 5′-AAAGCTAGCATGGACCGTCTGCTTGCCTC-3′ (sense) and 5′-CTCGAGTTGATCCAGTATTTTTTGGCGCC-3′ (antisense).

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