Ab291

Cells in supplemented EMM were collected and whole-cell protein extract was prepared by vortexing acid-washed glass beads in 20% trichloroacetic acid (TCA) and washing beads with 5% TCA. Lysates were boiled for 5 min in Laemmli Sample buffer (4% SDS, 60 mM Tris-HCl, pH 6.8, 5% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue) and analyzed by SDS-PAGE. Primary antibodies used were as follows: anti-phospho-H2A (Abcam ab17353; 1:1,000), anti-H2A (Cell Signaling 3636S; 1:1,000; re-probed after phospho-H2A), anti-GFP (Abcam ab291; 1:1,000), anti-myc (Abcam ab9106; 1:1,000) anti-PCNA (Santa Cruz sc-56; 1:1,000), and anti-cdc2 (Abcam ab5467; 1:1,000). After secondary antibody (Alexa Flour 488 or 647; 1:6,000) incubation, blots were developed using Amersham Typhoon biomolecular imager. Intensity of bands were quantified with ImageJ.

Densities of the 1-ml fractions from the flotation centrifugation analyses were determined from three independent experiments by measuring the weight of three 100 µl subfractions from each fraction. The fractions were precipitated with trichloroacetic acid (TCA) and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE gels were stained with Coomassie, Sudan black, or both and imaged using a ChemiDoc Touch imager (BioRad) or used for Western blotting. Polyclonal antibodies (pAb) against P12 (1:13,333; GeneCust) and phi6 (1:2500) and a monoclonal antibody against GFP (1:25,000; ab291, Abcam) were used as primary antibodies. HRP-labeled anti-rabbit pAb (1:125,000; Sigma Aldrich) or anti-mouse pAb (1:5000; PI-2000; Vector Laboratories Inc.) were used as secondary antibodies. Chemiluminescence was detected using a Western Lightning ECL Kit (Perkin-Elmer) and ChemiDoc Touch imager (BioRad) with 600 s of exposure time. Mass spectrometric analyses of P12 were performed in the Proteomics Unit of the University of Helsinki using liquid-chromatography–mass spectrometry × 2 (LC–MS/MS). Quantitative analyses of the EM thin sections was done using Aida Image Analyzer v 4.5.

Ede1 constructs were detected using an anti-GFP mouse monoclonal antibody (ab291, Abcam) at 1/2000 dilution, and an anti-Hog1 rabbit polyclonal antibody (sc-9079, Santa Cruz Biotechnology) at 1/1000 dilution was used as a loading control. Donkey antimouse IRDye 680 and anti-rabbit IRDye 800 secondary antibodies (926–68072 and 926–32213 respectively, LI-COR Biosciences) were used at a 1/10,000 dilution.