C terminal ddk tag

The SLC2A1-FAF1 and BCAS4-AURKA fusion open reading frames (ORFs) were PCR amplified from the index tumor (MPC10) and cloned into a mammalian expression vector pCMVentry, with a C-terminal DDK tag (OriGene, MD). Full-length expression constructs of FAF1, AURKA and RAE1 with DDK tags were obtained from OriGene. Expression of the fusion and wild-type protein constructs were detected with the anti-DDK monoclonal antibody 4C5 (Origene) by western blotting of 20μg of whole cell protein lysate as described[38 (link)]. Sequences of the ORFs are available at: http://rock.icr.ac.uk/Mackay/Micropapillary.

The SLC2A1-FAF1 and BCAS4-AURKA fusion open reading frames (ORFs) were PCR amplified from primary tumor MPC10 and cloned into a mammalian expression vector pCMVentry, with a C-terminal DDK tag (Origene). Cloned fusions, full-length 3-prime partner gene constructs or empty vectors were transfected into in four ER-positive breast cancer cell lines (MCF7, BT474, T47D and ZR75.1) using Lipofectamine 2000 (Invitrogen). Antibiotic selection was performed as previously described[38 (link)], and cell populations were assessed every 24 hours, for 9 days, using the CellTiter-Glo® cell viability assay (Promega)[38 (link)], with each reading normalized to day 1 to determine the fold change (Supplementary Methods).