Hydroxyurea synchronization of PCF cells expressing endogenous tagged TbCDC34-6HA.

<p>PCF 29–13 cells harboring pENT6B-TbCDC34-6HA were treated for 12 h with 0.2 mM hydroxyurea (HU), washed twice, and culturing was resume without HU. Samples were taken every two hours for flow cytometry and western blot analysis. (A) Histograms indicate results from cell sorting for DNA contents after cell cycle synchronization (propidium iodide channel). Dashed lines indicate the peak locations in the histograms from cell cycle. (B) Western blot showing protein levels of TbCDC34-6HA after HU release. Total proteins from 5x10⁶ cells were stained with anti-HA antibody (top) or anti-β Tubulin as a loading control (bottom). The loading buffer did not contain DTT. Arrow indicates the band corresponding to TbCDC34-6HA and the asterisk indicates the band corresponding to the modified version of the protein.