Xic2 is differentially phosphorylated in the absence and presence of single-stranded DNA. A. Xic2 phosphorylation shift assay. 35S-methionine labeled Xic2 was incubated in interphase egg extract (LSS or HSS) or mitotic extract (Δ90) as indicated in the absence (-) or presence of 80 or 200 units (U) of lambda phosphatase (λ-PPase). B. Xic2 phosphorylation shift and degradation assay. 35S-methionine labeled Xic2 was incubated in LSS with buffer (no DNA), single-stranded ΦX174 DNA (ssDNA, 10 ng/ul), pCS2+ plasmid DNA (dsDNA, 10 ng/ul), or XSC (10 ng/ul) at 23°C. Samples were analyzed by SDS-PAGE at 0-3 hrs. The mean percentage of remaining Xic2 from two independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. C. Xic2 degradation assay. 35S-methionine labeled Xic1 or Xic2 was incubated in HSS with (+) or without (-) single-stranded DNA (ssDNA) for 0–3 hrs as indicated. Molecular weight markers are shown in kilodaltons. D. Schematic representation of 35S-methionine labeled Xic2 phosphoforms in the absence of extract or DNA (IVT) (left lane), in the presence of LSS with or without XSC (middle lane), or in the presence of LSS or HSS with ssDNA (right lane). Unphosphorylated Xic2 is marked by the blue line (XIC2), Xic2 phosphoform 1 is marked by the purple line and the caret (>), and Xic2 phosphoforms 2 are marked by the pink lines and the asterisks (*). E. Xic2 co-immunoprecipitation with cyclin E. 35S-methionine labeled Xic2 was incubated in HSS without (no DNA) or with (ssDNA) as indicated. Xic2 was co-immunoprecipitated (IP) with anti-cyclin E or control antibody. 10% of the input reaction is shown (10% INPUT). In all figures, “XIC2-P” or the caret (<) and asterisks (*) indicate the phosphoforms of Xic2 and ubiquitinated Xic2 protein bands are indicated as “XIC2-(UB)n”.