Figure 2.
HOG pathway MEK Pbs2 forms a stable association with Hog1 in response to AA treatment. (A) Hog1 and Pbs2 associate weakly in the absence of stress. WT cells (DL3187) co-expressing Hog1-Myc (p3673) and Pbs2-HA (p3671) were grown in selective medium at the indicated pH. Anti-Hog1 IPs of extracts were separated by SDS-PAGE and subjected to immunoblot analysis. Molecular mass markers (in kDa) are shown on the right. (B) AA treatment induces association of Pbs2 with Hog1. WT cells (DL3187) co-expressing Hog1-Myc (p3673) and either Pbs2-HA (p3671) or Pbs2-DD (p3675) were treated with AA or not (C) and extracts were processed for co-IP, as above. (C) AA-induced association of Pbs2 with Hog1 is independent of the phosphorylation state of Pbs2. A sho1Δ ssk1Δ mutant (DL4306) transformed with a plasmid expressing either Pbs2-HA (p3671) or Pbs2-DD (p3675) was treated with AA and extracts processed for co-IP. (D) Time-course of AA-induced Pbs2 association with Hog1. WT cells (DL3187) co-expressing Hog1-Myc (p3673) and Pbs2-HA (p3671) were treated with AA for the indicated times. Extracts were processed for co-IP. The fold increase over signal from unstressed cells was calculated relative to the Hog1-Myc1 IP signal. (E) Time-course of AA-induced Hog1 activation. The samples in D were processed for immunoblot detection of Hog1 and P-Hog1. (F) Neither hyperosmotic stress nor arsenite treatment induces association of Pbs2 with Hog1. WT cells (DL3187) co-expressing Hog1-Myc (p3673) and Pbs2-HA (p3671) were treated with AA, arsenite (As; 1 mM for 10 min) or hyperosmotic stress with sorbitol (700 mM for 10 min). Extracts were processed for co-IP of Pbs2 with Hog1. (G) A hog1Δ strain (DL3158)-expressing Hog1-Myc (p3673) or Hog1-DADA-Myc (p3685). Source data are available for this figure: SourceData F2. Refer to the image caption for details.

HOG pathway MEK Pbs2 forms a stable association with Hog1 in response to AA treatment. (A) Hog1 and Pbs2 associate weakly in the absence of stress. WT cells (DL3187) co-expressing Hog1-Myc (p3673) and Pbs2-HA (p3671) were grown in selective medium at the indicated pH. Anti-Hog1 IPs of extracts were separated by SDS-PAGE and subjected to immunoblot analysis. Molecular mass markers (in kDa) are shown on the right. (B) AA treatment induces association of Pbs2 with Hog1. WT cells (DL3187) co-expressing Hog1-Myc (p3673) and either Pbs2-HA (p3671) or Pbs2-DD (p3675) were treated with AA or not (C) and extracts were processed for co-IP, as above. (C) AA-induced association of Pbs2 with Hog1 is independent of the phosphorylation state of Pbs2. A sho1Δ ssk1Δ mutant (DL4306) transformed with a plasmid expressing either Pbs2-HA (p3671) or Pbs2-DD (p3675) was treated with AA and extracts processed for co-IP. (D) Time-course of AA-induced Pbs2 association with Hog1. WT cells (DL3187) co-expressing Hog1-Myc (p3673) and Pbs2-HA (p3671) were treated with AA for the indicated times. Extracts were processed for co-IP. The fold increase over signal from unstressed cells was calculated relative to the Hog1-Myc1 IP signal. (E) Time-course of AA-induced Hog1 activation. The samples in D were processed for immunoblot detection of Hog1 and P-Hog1. (F) Neither hyperosmotic stress nor arsenite treatment induces association of Pbs2 with Hog1. WT cells (DL3187) co-expressing Hog1-Myc (p3673) and Pbs2-HA (p3671) were treated with AA, arsenite (As; 1 mM for 10 min) or hyperosmotic stress with sorbitol (700 mM for 10 min). Extracts were processed for co-IP of Pbs2 with Hog1. (G) A hog1Δ strain (DL3158)-expressing Hog1-Myc (p3673) or Hog1-DADA-Myc (p3685). Source data are available for this figure: SourceData F2.

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