α sic1

Protein extracts for Western blotting were prepared following cell fixation using trichloroacetic acid, as described [63] (link), and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Antibodies used for Western detection were, α-Clb2 (Santa Cruz, sc9071), α-Orc6 (clone SB49); α-Sic1 (Santa Cruz, sc50441), α-Tub1 (clone YOL1/34, AbD Serotec), α-HA (clone 12CA5), α-myc (clone 9E10), α-Pk (clone SV5-Pk1, AbD Serotec). Phos-tag was purchased from Wako Chemicals and added to SDS-polyacrylamide gels along with MnCl₂ according to the manufacturer's instructions.
For immunoprecipitation, cell extracts were prepared in EBXG buffer (50 mM HEPES pH 8.0, 100 mM KCl, 2.5 mM MgCl₂, 10% glycerol, 0.25% Triton X-100, 1 mM DTT, protease inhibitors) using glass bead breakage in a Multi Bead Shocker (Yasui Kikai). Extracts were precleared, incubated with antibody and finally adsorbed to Protein A Dynabeads. Beads were washed and elution was carried out in SDS-PAGE loading buffer. For the in vitro Nur1 dephosphorylation assay, immunoprecipitation was performed as above, then beads were resuspended in phosphatase buffer and 1 µg λ phosphatase (New England Biolabs), or 8 µg purified recombinant Cdc14 [4] (link), were added, followed by incubation at 30°C for 30 minutes before the reaction was stopped and proteins eluted by addition of SDS-PAGE loading buffer.