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. 2015 May 19;5:10243. doi: 10.1038/srep10243

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Copyright © 2015, Macmillan Publishers Limited

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(a) Diagram of the HA2-Tcd1 construct. Two HA epitopes were inserted after the initiation codon of TCD1. The neo3 cassette was inserted into the 5′ nontranscribed sequence. (b) Confirmation of complete replacement of endogenous TCD1 genes by HA-TCD1. Total genomic DNA isolated from HA-TCD1 and WT cells was digested with BglII and hybridized with the probe shown in (a) (c) Expression analysis of HA2-Tcd1. Total cell protein was prepared from the mating of HA-TCD1-B with HA-TCD1-C at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, and 24 h after mixing. The extracted protein was separated by SDS-PAGE. HA-Tcd1 was analyzed in Western blots with anti-HA antibody. (d) Viability of progeny. Results were obtained using the same method as in Fig. 2c.