α mouse hrp

Samples were resolved on 8% Tris/glycine polyacrylamide gels and transferred to a PDVF membrane (Millipore Immobilon, Merck, pore size 0.45 μm). The membranes were blocked by 5% non‐fat dry milk powder (NFDM) in TBS‐T (20 mM Tris–HCl pH 7.4, 150 mM NaCl and 0.1% (v/v) Tween‐20) for at least 1 h at RT. Primary antibodies were diluted in 5 % NFDM in TBS‐T as follows: α‐HA‐4D2 (Roche) 1:2,000; α‐PTH1R 4D2 (Thermo Fisher Scientific) 1:2,000; α‐FLAG‐HRP M2 (Sigma‐Aldrich) 1:5,000; α‐1D4‐HRP (Santa Cruz Biotechnology, sc‐57432) 1:2,000. Membranes were incubated for either 1 h at RT (α‐FLAG‐HRP) or overnight at 4°C with the primary antibody (α‐HA, α‐PTH1R and α‐1D4‐HRP), followed by 3 × 10 min washes in TBS‐T. Secondary antibodies, either α‐rat‐HRP (Cell Signaling) or α‐mouse‐HRP (Santa Cruz Biotechnology, sc‐516102), were used at 1:10,000 in 5% NFDM in TBS‐T for 1 h at RT followed by 3 × 10 min wash cycles in TBS‐T. Membranes were soaked in homemade ECL reagent (10 parts 0.1 M Tris–HCl pH 8.6 with 250 mg/l luminol, one part DMSO with 1,100 mg/l p‐hydroxycoumarin acid, and 0.003 parts 30% H₂O₂). After 1 min, signals were detected for 5 min in the dark (Gbox, Syngene).

α-Hsf1 (1:5000; Supplementary Fig. 4a and Supplementary Note 3), α-Sch9 (1:500; Supplementary Fig. 6a), α-Sse1 (1:10,000)^{9 (link)}, α-Ssb (1:5000)^{58 (link)}, α-Rpl35 (1:10,000)^{4 (link)}, α-Rpl17 (1:10,000)^{58 (link)}, α-Rpl19 (1:10,000)^{4 (link)}, α-Rps6 (1:10,000; Supplementary Fig. 6b), α-Rps9 (1:10,000)^{58 (link)}, α-Ssa (1:10,000)^{9 (link)}, α-Pgk1 (1:5000; Supplementary Fig. 6c), and α-Rps20 (1:10,000; Supplementary Fig. 6d) were rabbit polyclonal antibodies (Eurogentec, Rospert lab antibody collection). The rabbit polyclonal α-Maf1 (1:5000) was a kind gift from Olivier Lefebvre (Commissariat à l’Energie Atomique, France). α-phospho-Sch9-T570 (1:5000)^{24 (link)} was kindly provided by Robbie Loewith (University of Geneva). Commercially purchased antibodies were α-FLAG (1:10,000; Agilent 200471), α-GFP (1:5000; Life Technologies A11122), α-rabbit HRP (1:10,000; Pierce 31460) and α-mouse HRP (1:10,000; Santa Cruz SC-2005). Immunoblots were detected by enhanced chemiluminescence^{59 (link)}. Uncropped scans of the most important immunoblots are shown in Supplementary Figs 7–12.