The kit contains a rabbit polyclonal anti-phospho-PLK1 primary antibody, a goat anti-rabbit secondary antibody conjugated to the DyLight™ 549 Fluorophore, DAPI dye to identify the cell cycle phase by DNA content and various other reagents and buffers required for immunofluorescence labeling of cell cycle-specific PLK1 activation for high-content screening (HCS) assays.
Polo-like kinase 1 (PLK1) is a member of the serine/threonine protein kinase family and cdc5/polo subfamily, and it contains two polo box domains. PLK1 has multiple functions in the cell cycle, including cdc2/cyclin B regulation through phosphorylation of cdc25c phosphatase, centrosome maturation, spindle assembly, sister chromatin separation, cytokinesis and mitosis exit. Abundance, activity and localization of PLK1 are regulated during the cell cycle, and its proper subcellular localization is thought to be crucial for its function. Activation of PLK1 is controlled in part by phosphorylation during the cell cycle (highest in G2/M phase). Thr210 is the major phosphorylation site in activated PLK1 in mitotic cells and has been shown to interact with nuclear distribution gene C. PLK1 is associated with different mitotic structures including spindle poles, kinetochores, the midzone of the central spindle and the midbody in M phase of cell cycle. PLK1 is decreased and dephosphorylated at the end of mitosis. The activation of PLK1 can be monitored by the movement from the cytoplasm to the nucleus of phospho-PLK1 or the nuclear intensity change of PLK1 during the cell cycle. Depletion of PLK1 by siRNA knockdown induces apoptosis of cancer cells and inhibits cancer growth in vivo.
Figure 2. Phospho-PLK1 staining in asynchronous A549 lung carcinoma cells. Cells were fixed and stained according to the kit procedure with anti-phospho-PLK1 primary antibody, DyLight 549 Goat Anti-Rabbit Secondary Antibody (orange; rendered green) and DAPI Dye (blue). Phospho-PLK1 levels increase in the nucleus during progression of the cell cycle (arrows indicate cell cycle progression). The cell cycle phases were determined from the DNA content analysis of DAPI-stained cells. Phospho-PLK1 also stained the post-mitotic bridge in dividing cells.
Figure 3. Phospho-PLK1 activation in A549 cells treated with Nocodazole. DAPI staining of DNA content shown in blue; phospho-PLK1 staining with DyLight 549 (orange) Fluor shown in green. Panel A: non-treated cells. Panel B: cells treated with 0.5 μg/ml Nocodazole treatment for 16 hours.
Figure 4. Dose response curves of phospho-PLK1 for drugs that affect the cell cycle. Drugs were added to the cells (Vinblastine to HeLa cells, Nocodozale to A549 cells) at the indicated concentrations. Each data point represents eight wells from one to three separate plates. Phospho-PLK1 was measured using the output parameter of the nuclear fluorescence intensity (MEAN_CircTotalIntenCh2) with the Cellomics Compartmental Analysis Bioapplication. Values were normalized with the maximum control value and presented as % Control.EC50 Vinblastine = 11.0 nM. EC50 Nocodazole = 43.6 nM.
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