Fig. 3.

Chromatin association and phosphorylation of Rec8 is Scc3-dependent. a Cellular fractionation of starved and vegetative cells expressing Rec8-HAHis6 shows that the chromatin bound form is the slower migrating form. W: whole cell lysate, S: soluble fraction, C: chromatin bound fraction. In scc3i cells, Rec8 is not detected in the chromatin fraction, and only the faster migrating form is present in whole cells and the soluble fraction. Total protein loading is visualized in the Bio-Rad stain free gel prior to blotting. b Chromatin bound Rec8 is phosphorylated. Rec8-HAHis6 immunoprecipitated from the chromatin fraction of starved cells was treated with Lambda protein phosphatase (LPP). Both forms are present in the mock treated sample, but the phosphatase treated sample contains only the faster migrating form, indicating the modified form is phosphorylated. c Immunoprecipitation experiments were performed on soluble and chromatin fractions of Rec8-HA3His6 vegetative cells. The phosphorylated sites on Rec8 were identified by mass spectrometry analysis. Six residues show increased levels of phosphorylation in the chromatin fraction compared to the soluble fraction. The graphed values represent the ratio of the intensity of modified peptide to the intensity of unmodified counter peptide. Blue bars: chromatin fraction, Red bars: soluble fraction