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. 2015 Aug 11;12(8):824–837. doi: 10.1080/15476286.2015.1058476

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© 2015 The Author(s). Published with license by Taylor & Francis Group, LLC

© Georis Isabelle, Jennifer J Tate, Fabienne Vierendeels, Terrance G Cooper, and Evelyne Dubois

This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.

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Figure 7. — N-terminal tagging of GAT1 does not allow the detection of a truncated protein produced from the prematurely terminated transcript. Panel A. Scale representation of the GAT1 locus in the strains used in this figure. The GAT1 ORF is symbolized by a black arrow. C-terminal tagging cassettes are in orange and N-terminal tagging cassettes are in blue. The sites for transcription termination are indicated by a bold line. The sites for transcription initiation are indicated by a thin line. Panel B. Total RNA was isolated from gat1Δ (FV006), GAT1-HA³ (FV446), HA³-_(M40)GAT1-MYC¹³ (FV723) and HA³-_(M95)GAT1-MYC¹³ (FV726) cells grown in YNB medium with glutamine with (Rap) or without (Gln) rapamycin treatment. DAL5 mRNA levels were quantified by quantitative RT-PCR with primer pair DAL5_O1-O2. Panel C. Total protein extracts were prepared from P_GAL1-HA³-_(M40)GAT1 (FV685) and GAT1-HA³ (FV446) cells grown in YNB galactose medium with glutamine (Gln) or proline (Pro) as the nitrogen source, and subjected to Western blot analysis using anti-ha antibodies. Ponceau staining ensured proper loading and transfer efficiency (not shown). At regular exposure (like the upper panel, from 100kDa to 70kDa), no band was detected in the mass ranges under 70Kda. The lower panel (from 70kDa to 25kDa) is a higher exposure in the lower mass ranges. Panel D. Total protein extracts were prepared from wild type untagged (TB50), GAT1-MYC¹³ (FV063), GAT1-HA³ (FV446), HA³-_(M40)GAT1-MYC¹³ (FV723) and HA³-_(M95)GAT1-MYC¹³ (FV726) cells grown in YNB medium with glutamine (Gln) or proline (Pro) as the nitrogen source, and subjected to Western blot analysis using anti-ha or anti-myc antibodies. Pgk1 was used as the loading standard. Panel E. Total RNA was isolated from GAT1-MYC¹³ (FV063) and ski7Δ GAT1-MYC¹³ (FV739) cells grown in YNB medium with glutamine, ammonium or proline. GAT1 mRNA levels were quantified by quantitative RT-PCR with primer pairs GAT1_O1-O2.and GAT1_O3-O4.