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).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.