Fig. 4

S-acylation of Rif1 C466 and C473 in vivo. a Outline of the ABE protocol, including protein alkylation at free cysteines with NEM, removal of S-acyl groups including palmitoyl (Palm) using hydroxylamine (HA), and labeling with BMCC-biotin. Biotinylated proteins are captured on NeutrAvidin-coated beads and the presence of Rif1_NTD is analyzed by western blotting (WB). b Representative western blots of ABE assays performed with cells expressing Myc-tagged Rif1_NTD under control of a GAL1 promoter. Input: BMCC-biotin samples prior to biotin capture with and without HA. Input and AviF samples were probed with anti-Myc and anti-biotin antibodies as indicated. Fold enrichment of Rif1_NTD in AviF relative to wild-type is presented as mean values ± s.e.m. (n = 3 independent experiments). c Outline of the ACE protocol, including treatment of proteins with TCEP (reducing potential disulfide bridges between cysteine residues), alkylation at free cysteines with NEM, removal of S-acyl groups using DTT, and alkylation of freed-up cysteines with chloroacetamide. Myc-tagged Rif1_NTD is then immunoprecipitated for analysis by mass spectrometry (see Supplementary Fig. 4a for additional controls). d Mass-spectrometric analysis of tryptic Rif1 fragments spanning amino acids 463 to 479. Following ACE, Rif1_NTD tryptic peptides were subjected to parallel reaction monitoring (PRM), measuring NEM (unmodified Rif1) and/or CAM-labeled (reflecting in vivo S-acylation) C466 and C473 in wild-type vs. pfa4Δ. Integrated PRM counts are presented as mean values ± s.e.m. and were normalized using measurements of the five non-modified Rif1 peptides shown on the right (see Supplementary Table 1 for additional information). Data are shown in logarithmic scale (n = 3 independent experiments). e Measurements of C466/C473 NEM and/or CAM-labeled peptides (left panel) and unmodified control peptides (right panel) of Rif1 in untreated vs. Zeocin-treated wild-type cells. PRM analysis of Rif1_NTD peptides as in panel d. Mean values of integrated PRM counts ± s.e.m (n = 3 independent experiments) are shown in logarithmic scale. See Supplementary Fig. 4b, c for peptide transitions used in the experiment. Source data are provided as a Source Data file