Pbabe k ras 12v

cDNA encoding mCherry‐α‐tubulin, mCardinal‐H1, and dTomato‐VE‐cadherin were PCR‐amplified from pmCherry_α_tubulin_IRES_puro2 [kindly provided by Daniel Gerlich (Steigemann et al, 2009)], mCardinal‐H1‐10 [a gift from Michael Davidson (Chu et al, 2014; Addgene plasmid # 56161)], and tdTomato‐VE‐cadherin‐N‐10 (a gift from Michael Davidson (Addgene plasmid # 58142), respectively, and subsequently ligated into pMXs‐IRES‐Blasticidin (Cell Biolabs Inc.).
MCF10A ecoR cell lines allowing doxycycline‐inducible expression of EGFP‐NLP, EGFP‐CEP68 and EGFP‐PLK4 were described previously (Schnerch & Nigg, 2016). Doxycycline‐inducible MDCK II cells were generated using the same two‐step transduction strategy and enriched by antibiotic selection using hygromycin at 400 μg ml⁻¹ and puromycin at 2 μg ml⁻¹. Inducible MDCK and MCF10A cells stably expressing mCherry‐α‐tubulin, dTomato‐E‐cadherin and mCardinal H1 were generated by retroviral transduction and sorted by flow cytometry using BD FACSAria IIIu cell sorter (FACS Core Facility of Biozentrum). MCF10A ecoR cells stably expressing K‐Ras were obtained by ecotropic retroviral transduction with pBabe K‐Ras 12V [Addgene plasmid #12544, gift from Channing Der (Khosravi‐Far et al, 1996)].