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. 2014 Jul 9;42(13):8692–8704. doi: 10.1093/nar/gku597

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Interaction between She2 and Loc1 is important for efficient localization of ASH1 mRNA. (A). Diagram of Loc1-HA constructs (C1 to C5) cloned into the YCplac111 centromeric plasmid. (B) Co-immunoprecipitation assay to define the interacting domain of Loc1 with She2. Using monoclonal HA.11 antibody, Loc1-HA (C1 to C5) were immunoprecipitated and the presence of She2-myc was examined by western blotting using rabbit polyclonal anti-myc antibody. Upper panel shows input for each immunoprecipitation reaction. Middle panel shows western blot of co-immunoprecipitated She2-myc along with Loc1-HA (C1 to C5). Numbers at the bottom are quantification of the percentage of IP over Input. Values presented are mean ± SEM (N = 3). Bottom panel correspond to the same membrane stripped and hybridized with monoclonal HA.11 antibody to show the efficiency of Loc1-HA immunoprecipitation. Arrowheads indicate different Loc1-HA constructs. Asterisk indicates light chain of IgG. (C) Yeast genetic assay to assess ASH1 mRNA localization in Loc1 deletion mutants. K4452 loc1 strain was transformed by empty vector YCPlac111 or different Loc1-HA constructs (C1 to C5). Dilutions of exponentially growing yeasts were spotted on (–Leu) and (–Leu – Ade) plates and incubated at 30°C.