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HA-HACE1 transfection of osteosarcoma cells

AE Amal M El-Naggar
PC Paul W. Clarkson
GN Gian Luca Negri
BT Busra Turgu
FZ Fan Zhang
MA Michael S. Anglesio
PS Poul H. Sorensen
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Stable HACE1 re-expression in osteosarcoma cell lines was conducted using a retroviral system. HACE1 was tagged with haemagglutinin (HA) and transferred into an MSCV vector carrying resistance to hygromycin. The HECT domain cysteine residue, C876, critical for HACE1 E3 ligase activity30, was subjected to site-directed mutagenesis, converting cysteine-876 to serine (C876S) to create a non-functional control. Thus, three vectors were used, firstly a control MSCV empty vector, secondly an MSCV vector containing functional HA-HACE1 (HA-HACE1 vector), and thirdly an MSCV vector containing the putatively non-functional HA-C876S (HA-C876S vector). All vectors used in the current study were generated as previously described38.

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