JCS006502 Supplementary Material

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• Supplemental Table S1 -

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• Supplemental Figure S1 -

  Fig. S1. Pdd1-CFP is expressed at levels comparable to the endogenous protein. Whole cell extracts of PDD1-CFP transformants isolated 3, 6, 9 and 12 hours into conjugation, with (+) or without (-) CdCl2 induction, were fractionated on 4-20% SDS polyacrylamide gels, transferred to nitrocellulose and incubated with anti-Pdd1p antibodies (A) or anti-Histone H3, lysine four tri-methylated antibodies (H3K4me3) (B). The latter antibody served as a loading and normalization control between samples. Numbers to the left indicate the migration of Kaleidoscope protein size standards (Bio-Rad) in kDa. The migration of endogenous Pdd1p, Pdd1p-CFP and modified histone H3 are indicated to the right. Chemiluminescence was captured and quantified using a Fuji Film LAS-1000 imager and ImageGuage software. The abundance of Pdd1p-CFP (maroon bars) relative to endogenous Pdd1p (light blue bars) expression is shown in C. For each time point, the quantity of Pdd1p in uninduced cells was arbitrarily set to 1.0 and loading was normalized to total H3K4me3 chemiluminescence. The CFP-tagged Pdd1p levels never exceeded the levels of Pdd1p expressed from the native locus, but did add to the overall levels of Pdd1p in later time points.