α myc clone 9e10

At the indicated times, aliquots of the cultures were harvested and processed for ChIP of the cohesin complex as described [24 (link)]. Antibodies used for ChIP were α-Pk (clone SV5-Pk1, AbD Serotec), α-myc (clone 9E10) and α-HA (clone 12CA5). Processed chromatin immunoprecipitates and input DNA samples were hybridized to Affymetrix GeneChip S. cerevisiae Tiling 1.0R arrays. Presented is the genome-normalized ratio of the hybridization signals of the chromatin immunoprecipitate over the input DNA. Each bar in the bar graphs represents the average of 25 oligonucleotide probes within neighbouring 125 bp windows. For the line graphs, a smoothed moving average of 200–500 bp is shown. The microarray data are available from the GEO database under the accession number GSE80464. The quantitative analysis of cohesin binding to individual loci was performed as described, using previously described qPCR primer pairs [30 (link)], as well as primer pairs flanking the GAL2 locus, listed in the electronic supplementary material, table S2.

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.