Fig. 5. Localization and fractionation profile of endogenous hCAF1 over the course of the cell cycle. (A) Subcellular localization of endogenous hCAF1. HeLa cells were immunostained with anti-CAF1 polyclonal antibody. (B) MRC5 cells were synchronized by serum starvation (t0). After re-addition of serum, cells were analyzed at indicated time points for either immunofluorescence or fluorescence-activated cell sorting (FACS) analysis. (B1) Cell-cycle distribution, as determined by FACS analysis. The cells were fixed in 70% ethanol and treated with RNase. The DNA was stained with propidium iodide. (B2) Immunofluorescence of endogenous hCAF1. MRC5 cells were fixed in 4% PAF at the times indicated and stained for detection of endogenous hCAF1 with polyclonal antibody anti-hCAF1, followed by fluoresceine isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody. (C) Distinct steady-state fractionation profiles of hCAF1 during the course of the cell cycle. Lysates of MRC5 cells arrested in G0 by serum starvation or in G1-S 17 hours after serum restimulation were fractionated on a Superose 6 column (cell lysates and Superose 6 column preparations are described in Fig. 1A). The elution profiles of endogenous hCAF1 were analyzed by western analysis using anti-hCAF1-specific antibody.