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

Figure 3. hPaf1 is ubiquitinylated and degraded at the metaphase/anaphase transition.

Figure 3

Panc1 cells were grown at low density on cover slips for 24 h, and arrested at G1/S (T0) and G2/M (N0) boundaries by double thymidine and thymidine/nocodazole blocks, respectively. Acetone/methanol (1∶1)-fixed Panc1 cells were labeled using a FITC-conjugated anti-hPaf1 monoclonal antibody and counterstained by propidium iodide. Up to 10 fields were observed by confocal microscopy for cells after 12 h of double thymidine treatment release (T+12) and compared with 10 fields of thymidine/nocodazole treated cells just before release (N0). Representative pictures are presented in A). The black arrows indicate the inter-chromatine accumulation of hPaf1 in the prophase for the cells in N0; accumulation was not detected at T+12. B) Proteins from nuclear extracts performed at T0, T+8, N0, and S0 (shake off cells in N0) were immunoprecipitated using the rabbit polyclonal antibody anti-ubiquitin (Calbiochem), and immunoblotted with anti-hPaf1 antibody. An aliquot of the nuclear protein extracts was loaded as a control for hPaf1 expression (input), and immunoprecipitation was carried out using an isotype IgG control antibody as a negative control. C) hPaf1 degradation by the proteasome was confirmed using the proteasome inhibitor MG132. After double thymidine block, the cells were released either in ethanol control or MG132 (20 µM) for 12 h. Protein lysates were imunoprecipitated using the rabbit polyclonal antibody anti-ubiquitin (Calbiochem) and immunoblotted with anti-hPaf1 antibody. D) Panc1, CD18/HPAF, and human 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 hPaf1 expression and sub cellular localization by confocal. In the prophase, hPaf1 was found aligned on filament-like structures (D3) to be concentrated at the pole in the metaphase (D1, D2: black arrow). The Z-section (D4, D5, and D6) revealed that in the metaphase, hPaf1 formed a crown at both poles of the cells, potentially surrounding the centrosomes (white arrow). E) CD18/HPAF cells were arrested at the G1/S boundary and released for 12 h to reach a maximum of mitosis. The cells were analyzed for hPaf1 and tubulin expression and subcellular localization by confocal. In metaphase, hPaf1 co-localized with tubulin, confirming the centrosomal localization of hPaf1.