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. 2009 Sep 22;4(9):e7077. doi: 10.1371/journal.pone.0007077

Figure 6. hPaf1 during initiation of DNA replication and spindle formation.

Figure 6

A) Protein extracts from G1/S (double thymidine arrested: T0) and G2/M (thymidine/nocodazole arrested: N0) were prepared, and equal amounts of proteins were used for immunoprecipitation using HP180-12 and SJK132-20 mouse monoclonal anti-Polymerase α antibodies. Immunoprecipitates were immunoblotted with anti-hPaf1 and anti-hLeo1 antibodies. An aliquot of the protein extracts at T0 was loaded as the control expression (input). B) Fibroblast cells were arrested at the G1/S boundary and released for 12 h to reach a maximum of mitosis. The cells were analyzed for Pol α expression and sub cellular localization by confocal using HP180-12 antibody 2 h after release. At that time frame, cells invalidate for hPaf1 expression presented Pol α loaded to chromosomal DNA while cytoplasmic for the control cells. C) Invalidation of hPaf1 was evaluated by confocal microscopy. The nuclear localization of hPaf1 stained in blue using the Cy5 dye was detected for the MCF7 cells transfected by the scramble siRNA, while no staining was detected for the cells transfected with hPaf1 siRNA. D) Twenty randomly chosen cells in metaphase were observed on each slide by confocal microscopy using a FITC-labelled anti-tubulin monoclonal antibody (DM1A, Abcam). Representative pictures for MCF7 control and MCF7 invalidated for hPaf1 are presented. Eighty percent of the cells observed presented defect in tubulin polymerization and spindle formation.