Anti ha antibody f 7

After electrophoresis (SDS–PAGE, or Phos‐tag SDS–PAGE), proteins were transferred onto Amersham™ Protran™ 0.45 μm nitrocellulose membranes (GE Healthcare) or onto Amersham™ Hybond ™ P 0.45 μm PVDF membranes (GE Healthcare). Phosphorylated Hog1 was detected by immunoblotting using the anti‐phospho‐p38 MAPK (T180/Y182) antibody #9211 (Cell Signaling Technology). Hog1 was detected using the anti‐Hog1 antibody yC‐20 (Santa Cruz Biotechnology). FLAG‐Hog1 was detected by anti‐FLAG M2 antibody (Sigma‐Aldrich). Pbs2‐HA was detected by anti‐HA antibody F‐7 (Santa Cruz Biotechnology). Phosphorylation of Pbs2 Thr‐518 was detected by polyclonal anti‐Pbs2 phospho‐T518 antibody custom produced by SCRUM Inc. Phosphorylated Kss1 and Fus3 were detected using the anti‐phospho‐p44/42 MAPK (Erk1/2; T202/Y204) Rabbit monoclonal antibody #4370 (Cell Signaling Technology).

Strains were grown in the indicated conditions and cross-linking was performed as described previously[42 (link)]. Press-dried mycelia was frozen in liquid nitrogen and stored at -80˚C until chromatin preparation, which was performed as described previously[43 (link)]. For immunoprecipitation, 2 ug of anti-HA antibody (F7, Santa Cruz) was used. Library preparation was carried out according to[44 (link)] with the exception of the first end-repair step, which was replaced by the NEBNext End-Repair module (Cat. no. E6050). Libraries were checked and concentration determined using DNA High Sensitivity Bioanalyzer assay, mixed in equal molar ratio as described[44 (link)] and subjected to sequencing using Illumina HiSeq2500.

293 T cells were transduced with vectors as indicated with HA-tagged ubiquitin constructs for 48 h. Before harvesting, cells were treated with 10 μM MG132 for 24 h (MedChemExpress, Cat No. HY-13259). The cells were then lysed in Pierce IP lysis buffer containing a protease inhibitor cocktail and immunoprecipitated with anti-Flag magnetic beads. Ubiquitination was detected by using an anti-HA (F-7) antibody (Santa Cruz, Cat No. sc-7392).