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. 2015 Jun 1;26(11):2128–2138. doi: 10.1091/mbc.E14-11-1552

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© 2015 Herrador et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — Effect of Rim21 C-tail overexpression on RIM signaling and Rim8 localization and phosphorylation. (A) Phenotypic analysis of rim mutant strains overexpressing Rim21 or Dfg16 C-tails (CT). Y01150 (rim21∆), Y11806 (dfg16∆), Y06196 (rim9∆), and Y04414 (rim8∆) were transformed with pADH1-Flag-Rim21CT, pADH1-Flag-Dfg16CT, or empty vector (none). Growth tests in the presence of LiCl were as described for Figure 1D. Immunoblot analysis of transformants expressing Rim21CT and Dfg16CT confirmed the expression of both proteins (unpublished data). (B) Effect of Rim21 C-tail overexpression on Rim101 processing. OVY24 (wild type [WT] RIM101-HA) and OVY43 (rim21∆ RIM101-HA) were transformed with pADH1-Flag-Rim21CT (+) or empty vector (–). After a shift to pH 7.5, protein extracts were prepared and immunoblotted with anti-HA antibody. (C) Effect of ambient pH on Rim8 localization. OVY172 (RIM8-3GFP) was grown to mid log phase in YPD medium and then injected into a microfluidics device in SD medium containing 0.1 M HEPES, pH 4. Cells were then perfused with SD medium containing 0.1 M HEPES, pH 7, and examined by fluorescence and DIC microscopy before and after the change of medium at the indicated time. (D) Rim8 localization in cells overexpressing Rim21 C-tail. OVY172 (RIM8-3GFP) and OVY304 (rim21∆ RIM8-3GFP) were transformed with pADH1-Flag-Rim21CT or empty vector as indicated on the left. Cells were grown to mid log phase on SD medium (final pH 3.5) and examined by fluorescence and DIC microscopy. (E) Colocalization of Rim8 and overexpressed Rim21 C-tail. OVY172 (RIM8-3GFP) and OVY31(rim8∆) were transformed with pADH1-RFP-Rim21CT, grown to mid log phase on SD medium (final pH 3.5), and examined by fluorescence and DIC microscopy. (F) Effect of Rim21 C-tail overexpression on Rim8 phosphorylation. Y04414 was cotransformed with pHA-Rim8 (top) or a Rim8 truncated derivative (residues 1–460; bottom) and pADH1-Flag-Rim21CT (+) or empty vector (–). Transformants were grown to mid log phase (final pH 3.5), and protein extracts were prepared and immunoblotted with anti-HA antibody.