Figure 6. Ablation of SG components... | Rockefeller University Press

Figure 6.

$Ablation of SG components blocks AA-induced activation of Hog1 and its association with Pbs2. (A) WT cells (DL3187), a sho1Δ ssk1Δ mutant (DL4306), and the quintuple deletion mutant (sam1Δ sam2Δ jlp1Δ pdc6Δ met5Δ; Quint Δ; DL4622) co-expressing Hog1-GFP (p3680; open bars) and Pbs2-mCherry (p3686; gray bars) were treated with AA and SGs that were isolated from cell lysates. SG preparations were assessed by fluorescence microscopy for quantitative analysis of Hog1-GFP and Pbs2-mCherry granules and co-localization (Co; black bars) of these signals. Values represent the mean number of granules detected per visual field (n = 3) ± standard deviation. (B and C) WT cells (DL3187) or the indicated deletion mutants were treated with AA for 10 min or untreated (Con). Extracts were processed for immunoblot detection of Hog1 and P-Hog1. (D) WT cells (DL3187) and the quintuple mutant (DL4622) were subjected to hyperosmotic stress with the indicated concentration of NaCl for 5 min and extracts were treated as above. (E) The strains in D, co-expressing Hog1-Myc (p3673) and Pbs2-HA (p3671), were treated with AA or untreated (C) and anti-Hog1 IPs of extracts were separated by SDS-PAGE and subjected to immunoblot analysis for co-IP of Pbs2-HA. (F) AA-induced Phospho-Hog1 is largely restricted to SGs. SGs were prepared from lysates of WT cells (DL3187) treated with AA or untreated (Con). The SG fraction (25% of total) and the depleted cytoplasm (0.5% of total) were processed for immunoblot detection of Hog1, P-Hog1, and Pab1. A shorter exposure of the Hog1 signals is included. Molecular mass markers (in kDa) for Pab1 are on the right. Source data are available for this figure: SourceData F6. Refer to the image caption for details.$

Ablation of SG components blocks AA-induced activation of Hog1 and its association with Pbs2. (A) WT cells (DL3187), a sho1Δ ssk1Δ mutant (DL4306), and the quintuple deletion mutant (sam1Δ sam2Δ jlp1Δ pdc6Δ met5Δ; Quint Δ; DL4622) co-expressing Hog1-GFP (p3680; open bars) and Pbs2-mCherry (p3686; gray bars) were treated with AA and SGs that were isolated from cell lysates. SG preparations were assessed by fluorescence microscopy for quantitative analysis of Hog1-GFP and Pbs2-mCherry granules and co-localization (Co; black bars) of these signals. Values represent the mean number of granules detected per visual field (n = 3) ± standard deviation. (B and C) WT cells (DL3187) or the indicated deletion mutants were treated with AA for 10 min or untreated (Con). Extracts were processed for immunoblot detection of Hog1 and P-Hog1. (D) WT cells (DL3187) and the quintuple mutant (DL4622) were subjected to hyperosmotic stress with the indicated concentration of NaCl for 5 min and extracts were treated as above. (E) The strains in D, co-expressing Hog1-Myc (p3673) and Pbs2-HA (p3671), were treated with AA or untreated (C) and anti-Hog1 IPs of extracts were separated by SDS-PAGE and subjected to immunoblot analysis for co-IP of Pbs2-HA. (F) AA-induced Phospho-Hog1 is largely restricted to SGs. SGs were prepared from lysates of WT cells (DL3187) treated with AA or untreated (Con). The SG fraction (25% of total) and the depleted cytoplasm (0.5% of total) were processed for immunoblot detection of Hog1, P-Hog1, and Pab1. A shorter exposure of the Hog1 signals is included. Molecular mass markers (in kDa) for Pab1 are on the right. Source data are available for this figure: SourceData F6.