Fig. 1. Cell-cycle-dependent chromatin-binding of proteins in A39 cells. Cell cycle phases of logarithmically growing A39 cells were separated by centrifugal elutriation. The DNA content of the different fractions was determined by FACS. Soluble and chromatin-bound proteins of each fraction were separated by cell fractionation and investigated after SDS-PAGE by immunoblot analysis. (A) FACS profiles of the separated fractions (top) and immunoblot analysis of chromatin-associated cyclins A, B1 and E (bottom). (B) Immunoblot studies of EBNA1 and pre-RC components using antibodies as indicated on the right. Chromatin-binding experiments of hsOrc1 were performed in parallel in the presence or absence of 25 µM MG132 as indicated on the left. (Bottom) To analyse the relevance of a 26S-proteasome-dependent degradation, whole cell extracts (WCE) were prepared by lysing cells in RIPA buffer in the absence (left) or presence (right) of 25 µM MG132. After SDS-PAGE of equivalent amounts of WCE, the presence of HsOrc1p was detected by immunoblotting. (C) Relative ratios of chromatin bound proteins analysed in (B). Signal intensities of the respective autoradiograph were quantified using NIH Image and plotted against the flow rate (ml minute^-1) corresponding to cell cycle progression. The highest intensity of each individual factor was set to 100%. HsOrc4p and HsOrc6p are not shown for the clarity of the figure. (D) For G0 experiments, A39 cells were grown to high density and arrested for 3 days. G0-arrested and logarithmically growing A39 cells (FACS profiles on the top left) were fractionated using the chromatin-binding protocol. Soluble (S) and chromatin-bound (Ch) proteins were separated by SDS-PAGE and immunoblots were probed with antibodies as indicated on the right.