Fig 1: Cdk4/6 induction synchrony can reveal transient cell cycle events. hTERT-RPE1 cells were grown to around 1.5 × 104 cells cm-2 in DMEM (+10% serum), trypsinized and plated at 4.4 × 103 cm-2 in 10 cm dishes. Twelve hours later, 150 nM palbociclib was added. Twenty-four hours after palbociclib addition, the cells were washed twice in growth medium before the medium was replaced with pre-warmed DMEM (+10% serum) that did not contain any palbociclib. One 10 cm dish was taken for each sample every hour to generate the propidium iodide FACS profiles in 13 h batches (a) to gauge the fluctuations in 2 N DNA content in the population (b), while sampling to monitor the indicated markers by western blot every 2 h (c). The numbers next to the plots in (a) indicate hours since release with U indicating an untreated control population. Both the mitotic kinesin 5 motor protein Eg5 and phosphorylation of the serine of histone H3 at position 10 peak as cells return from the 4 N state to the 2 N state (mitosis and cell division). This plot shows one of the three repeat experiments, which revealed similar fluctuations in the same cell cycle markers.
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