Anti atf1

For immunoprecipitation of Flag-tagged Pcr1 protein, S. pombe strains expressing Pcr1-Flag and/or Flp1-HA fusion proteins were grown in YES at 30 °C to an OD₆₀₀ of 0.5. Then H₂O₂ (1 mM) was added to part of the cultures and after 10 min incubation formaldehyde (1.5% vol/vol) was added for 30 min at room temperature. Cells were pelleted and washed three times with ice cold TBS. Pellets were resuspended in 500 ml lysis buffer (20 mM Tris, pH 8, 100 mM NaCl, 2 mM EDTA and 0.5% NP-40) supplemented with a protease inhibitor cocktail (Complete, EDTA-free, Sigma-Aldrich) and disrupted with Glass-Beads in a Fast-Prep. Cell lysates were clarified by centrifugation at 13.000 rpms and protein concentration was determined using BCA Protein Assay kit (Pierce Chemical, Rockford, IL). Cell extracts (3 mg) were incubated with dynabeads protein G (Invitrogen) bound to monoclonal anti-Flag antibody (Agilent Technologies) for 5 h at 4 °C. Beads were washed four times with lysis buffer and resuspended in loading buffer. Immunoprecipitates were resolved by SDS-PAGE, transferred to Nitrocellulose membranes using a Bio-Rad transfer unit and analyzed with anti-HA-HRP conjugated (Miltenyi Biotec), anti-Flag-HRP conjugated (Sigma) and anti-Atf1 (Abcam) antibodies.

Whole cell extracts were prepared by precipitation with trichloroacetic acid (TCA). S. pombe strains were grown in YES medium to OD₆₀₀ of 0.4–0.5 and cells (5 ml) were collected by centrifugation just after the addition of 100% TCA to a final concentration of 10% and washed in 20% TCA. Cell disruption was performed with Glass-Beads in a Fast-Prep and 12.5% TCA. Cell lysates were pelleted by centrifugation at 3.000 rpms and resuspended in 1X LB loading buffer and Tris base. Samples were electrophoretically resolved by SDS-PAGE and transferred to Nitrocellulose membranes using a Bio-Rad transfer unit. Blots were then probed against antibodies indicated. Antibodies used: monoclonal anti-Atf1 (Abcam), anti-HA-HRP conjugated (Miltenyi Biotec), anti-Flag-HRP conjugated (Sigma), anti-Myc-HRP conjugated (Miltenyi Biotec), anti phospho-p38 MAP Kinase (Cell Signaling) and anti-α-tubulin (Sigma-Aldrich).