Figure 2. Localization of hPaf1 during the cell cycle in Panc1 cells.

hPaf1 expression was monitored in a synchronously growing population of Panc1 cells. Panc1 cells were arrested at the G1/S boundary by double thymidine block, then released in normal medium containing serum (time 0 = T0) and analyzed every 2 h for 24 h by flow cytometry (after propidium iodide staining), confocal microscopy, and Western blotting of cytoplasmic and nuclear cell fractions. A) Eighty percent of the cells were blocked at the G1/S boundary. After release, the cells progressed into the cell cycle synchronously to reach a peak of mitosis after 10–12 h. Representative fields from the confocal analysis are presented in B). The cells were grown at low density on cover slips for 24 h, and arrested at the G1/S boundary by double thymidine incorporation. The acetone/methanol (1∶1)-fixed Panc1 cells were labeled using a FITC-conjugated anti-hPaf1 monoclonal antibody and counterstained by propidium iodide. The amount of fluorescence detected for the cells in T+12 (G2/M) was twice the level detected in T0 (G1/S). Up to 10 fields were monitored for each phase of the cell cycle. Cells in the prophase, metaphase, anaphase, and telophase showed very low to no expression of hPaf1. C) Nuclear and cytoplasmic protein extracts were prepared every 2 h and analyzed by immunoblotting using anti-hPaf1 and anti-hLeo1 antibodies. The JLA-20 antibody recognizing β actin was used as a loading control.