Xic2 is ubiquitinated and degraded in a DNA dependent manner. A. Degradation assay. 35S-methionine labeled Xic1, Xic2, and Xic3 were incubated in Xenopus interphase egg extract (Low Speed Supernatant, LSS) in the absence (-) or presence (+) of Xenopus sperm chromatin (XSC) for 0 to 3 hours as indicated. The mean percentage of remaining protein from two independent experiments is shown (% protein remaining) where the zero hour time point was normalized to 100%. Xic1, Xic2, and Xic3 protein bands are marked on the right including ubiquitinated [(UB)n] forms of Xic2. B. Xic2 ubiquitination assay. 35S-methionine labeled Xic2 was incubated in LSS with methyl ubiquitin (3 mg/ml) to stabilize the ubiquitinated species in the absence (-) or presence (+) of XSC for 0 to 3 hours as indicated. Xic2 protein bands are marked on the right including ubiquitinated [(UB)n] forms. C. Nuclei spin down assay. Nuclei spin down assay was employed to separate cytosolic (CYT) and nuclear fractions (NUC) after incubation of [35S]-methionine labeled Xic2 with LSS containing XSC. The input (INPUT) represents 1/15th of the sample before centrifugation and the cytosolic (CYT) represents 1/15th of the cytosolic fraction after centrifugation. Xic2-(UB) n denotes ubiquitinated Xic2. D. Xic2 immunoblot. Left panel: Xenopus LSS or XTC cell extracts were immunoprecipitated (IP) using anti-Xic2 (XIC2) or normal rabbit serum (Mock) antibody and then immunoblotted with anti-Xic2 antibody. Ten percent of the immunoprecipitation reaction was loaded directly (10% INPUT). Right panel: XTC cells were treated with gamma irradiation (IR, 10 Gy) and harvested 4 or 8 hrs following treatment. Lysates were then examined by immunoblotting with anti-Xic2 antibody. For all figures, the molecular weight marker (M) is shown in kilodaltons.