Ppz phosphatases regulate specific phosphorylation events within the Rsp5 adaptor network. (A) SILAC-based quantitation of the FLAG-Rsp5 interaction network in WT (H) and ppz mutant (L) cells. Experiment was performed as shown in Fig. S1 A using FLAG-Rsp5 as the bait. Normalized H:L ratios were measured and averaged over multiple biological replicates (n = 3). Error bars indicate standard deviation of measurements on peptides from at least two experiments. Immunoblot (inset) shows recovery (1%) of FLAG-Rsp5 as bait in these samples. Additional data for this experiment are shown in Fig. S4. (B) SILAC-based quantification was performed on Rsp5 phosphopeptides enriched by immobilized metal affinity chromatography. A schematic (top) illustrates the phosphorylation events detected and quantified relative to known domain organization. Color coding of domain organization applies to the graph depicting Rsp5 phosphopeptide quantification (bottom panel). (C–E) SILAC-based quantification of phosphorylation events resolved and quantified in the Rsp5 adaptor network, including Art3/Aly2 (C), Art4/Rod1 (D), and Art6/Aly1 (E). For all panels in Figure 6, normalized H:L ratios were measured and averaged over multiple biological replicates (n = 3). Error bars indicate standard deviation of measurements on peptides from at least two experiments (n ≥ 2). Schematic representations of each adaptor are shown at the left, and phosphopeptide quantification is shown on the right. Additional phosphoprofiling data from these experiments are shown in Fig. S4.