Fig. 3

a Heatmap showing SILAC ratios of selected phosphopeptides at 0, 5, 15 and 30 min after treatment with 0.5 M NaCl. Hallmarks: well-known phosphorylation events of osmostress signaling. Indirect targets: stress-inducible and inhibitor-susceptible phosphopeptides phosphorylated at non–S/T-P motif sequences [4]. Promiscuous p-sites: phosphorylation sites targeted by multiple kinases. b and c Average stress-induced phosphorylation kinetics of Hog1-dependent (above) and Hog1-independent (below) phosphorylation sites in a wild type and hog1Δ strain. d-g Illustration of PRM-measured phosphorylation patterns for Hog1-dependent and -independent sites upon hyperosmotic stress (+ 0.5 M NaCl) and inhibitor treatment (SPP86). N (biological replicates) = 3. For a given phosphorylation site, the green box plots represent the (mean) normalized intensities for the respective phosphopeptide(s). The yellow box plots illustrate the normalized intensities for unphosphorylated counter-peptides. Significance was assessed by comparing intensities derived from all pooled inhibitor-treated samples with those from the mock sample (t-test, p < 0.05). h Above: Representative Western blot showing M-track protein protein proximity signals obtained for Kic1. Hog1-protA-H3: background control, Nup2: positive control. Below: Proximity signals. n = 3 replicates per sample except when indicated differently. Ratios are log₂-transformed. Black lines indicate average proximity signal. Proximity signals that differ significantly from background are marked in green (q ≤ 0.01) and orange (q ≤ 0.05 and > 0.01) filled circles. Grey filled triangles: q > 0.05. ^N: N-terminal HKMTmyc fusion. i and j Newly identified Hog1 network hubs based on STRING. Red filled circles: putative target proteins identified in this study. ^M: positive M-track signal. Gray circles: first neighbor according to STRING. Shaded circles enclosing groups of proteins highlight functional groups. Filled lines indicate high, dashed lines confidence score ≤ 0.4 according to STRING