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. 2018 Dec 1;32(23-24):1576–1590. doi: 10.1101/gad.318709.118

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© 2018 Leskoske et al.; Published by Cold Spring Harbor Laboratory Press

This article, published in Genes & Development, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.

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Figure 5. — Phosphorylation at its MAPK sites displaces Avo2 from the PM. (A) Strains yAEA348 (Avo2^WT-mNG-3xHA Pil1-RFP), yAEA349 (Avo2^9A-mNG-3xHA Pil1-RFP), and yAEA350 (Avo2^9E-mNG-3xHA Pil1-RFP) were grown to mid-exponential phase and examined by fluorescence microscopy as described in the Materials and Methods and processed using CellProfiler (as in Supplemental Fig. S3). (B) Mean values of the relative pixel intensities of the Pil1-RFP and Avo2-mNG foci in images, as in A, were measured using CellProfiler, and those obtained for strain yAEA348 (Avo2^WT-mNG-3xHA Pil1-RFP) were set to 100%, to which all of the other values were normalized. Error bars represent 95% confidence intervals. (**) P < 0.0001. (C) The same cells as in A were resolved by standard SDS-PAGE and analyzed by immunoblotting. A representative experiment is shown. Values below the lanes represent the relative Avo2 to Pgk1 ratio (average of three independent experiments with SEM). (D) The mean pixel intensity of the Avo2-mNG foci that colocalized with Pil1-RFP was measured using CellProfiler as in B. (**) P < 0.0001. (E) The mean pixel intensity of the Avo2-mNG foci that did not colocalize with Pil1-RFP was measured using CellProfiler as in B. (**) P < 0.0001. (F) Strains BY4741, YFR589 (Avo2-mKate Tor2-mNG-3xHA), YFR624 (Avo2-mKate Avo3-3xFlag Tor2-mNG-3xHA), YFR626 (Avo2^9A-mKate Avo3-3xFlag Tor2-mNG-3xHA), and YFR628 (Avo2^9E-mKate Avo3-3xFlag Tor2-mNG-3xHA) were propagated in YPD, harvested, and lysed, and equal amounts of the resulting extracts were immunoprecipitated with anti-Flag antibody. Proteins in the input extracts and in the immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting. A representative experiment is shown. Values below the right lanes represent the relative Avo2 to Tor2 ratio (average of three independent experiments with SEM), with the value obtained for strain YFR624 (Avo2-mKate Avo3-3xFlag Tor2-mNG-3xHA) set as 1.00. (G) Model showing that under conditions that damage cell wall structure, activation of the CWI pathway exerts feedback that will negatively regulate the growth-promoting functions of TORC2.