Total cell extracts were prepared by trichloroacetic acid (TCA) precipitation from 5-ml aliquots of sporulation cultures as previously described (13 (link)). Analysis of Mek1 phosphorylation using Phos-tag gels was performed as reported (8 (link)). The antibodies used are listed in Supplementary Table S3. The ECL or ECL2 reagents (ThermoFisher Scientific) were used for detection. The signal was captured on films and/or with a ChemiDoc XRS system (Bio-Rad) and quantified with the Quantity One software (Bio-Rad).
For immunoprecipitation of Pch2, 5-ml aliquots from 16 h meiotic cultures were crosslinked with 1% formaldehyde for 10 min at 30°C. The reaction was quenched by adding glycine to 250 mM and incubating for 5 min on ice. Cells were collected, washed and broken with glass beads in lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris HCl pH 8.0) containing protease inhibitors (Complete Ultra Tablets, Roche). Clarified extracts were immunoprecipitated with anti-HA antibodies conjugated with magnetic MicroBeads using the μMACS Epitope Tag Protein Isolation Kit (Miltenyi Biotec) following the manufacturer's protocol.
For immunoprecipitation of Pch2, 5-ml aliquots from 16 h meiotic cultures were crosslinked with 1% formaldehyde for 10 min at 30°C. The reaction was quenched by adding glycine to 250 mM and incubating for 5 min on ice. Cells were collected, washed and broken with glass beads in lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris HCl pH 8.0) containing protease inhibitors (Complete Ultra Tablets, Roche). Clarified extracts were immunoprecipitated with anti-HA antibodies conjugated with magnetic MicroBeads using the μMACS Epitope Tag Protein Isolation Kit (Miltenyi Biotec) following the manufacturer's protocol.
