After 48 h, whole cell lysates were separated by SDS-PAGE and probed with indicated antibodies

After 48 h, whole cell lysates were separated by SDS-PAGE and probed with indicated antibodies. conversation with the PP6 complex. Finally, we find that PLK3 is usually phosphorylated at Thr219 in the T-loop and that PP6 constantly dephosphorylates this residue. However, in contrast to PLK1, phosphorylation of Thr219 does not upregulate enzymatic activity of PLK3, suggesting that activation of both kinases is usually regulated by distinct mechanisms. mRNA is usually presented as the ratio to mRNA. 2.5. Immunofluorescence Cells grown on coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.5% Triton X1?00 for 10 min. Cells were further incubated with ice-cold methanol for 5 min and blocked with 3% BSA in PBS for 30 min. Coverslips were incubated with primary antibodies for 3 h, washed with PBS, and incubated with AlexaFluor-conjugated secondary antibodies for 1 h. Mounting was performed using Vectashield. Imaging was performed using Leica Sp8 confocal microscope equipped with 63 oil objective (NA 1.40). Images were analyzed using LAS AF Lite software (Leica, Wetzlar, Germany). Induction of DNA damage response was evaluated as described previously [32]. Briefly, cells were exposed to ionizing radiation (3 Gy) using X-RAD 225XL instrument (Precision; Cu filter 0.5 mm), fixed with 4% PFA, permeabilized with 0.5% Triton X1?00, and probed with antibody against H2AX (Cell Signaling Technology). Images were acquired using Olympus ScanR system equipped with 40/NA 1.3 objective (Olympus, Tokio, JApan). Number of H2AX-positive foci per nucleus was decided using spot detection module. More than 300 nuclei were quantified per condition. 2.6. Immunoprecipitation HEK293 cells stably expressing EGFP or EGFP-PLK3 were extracted by IP buffer (20 mM HEPES pH 7.5, 10% glycerol, 150 mM NaCl, 0.5% NP40) supplemented with cOmplete protease and PhosSTOP phosphatase inhibitors (Sigma) and sonicated for 3 20 s on ice. Cell extracts were cleared by centrifugation at 15,000 rpm 10 min at 4 C and incubated with GFP-Trap beads (Chromotek, Planegg, Germany) for 2 h. After three washes in IP buffer, bound proteins were eluted from beads by Laemli buffer and analyzed by immunoblotting. Alternatively, bound proteins were analyzed by mass spectrometry using Orbitrap Fusion (Thermo Scientific). Proteins bound to EGFP-PLK3 that were enriched compared to the empty EGFP control in at least two out of three impartial experiments were considered as potential interactors and were validated by immunoprecipitation followed by immunoblotting. For in vitro kinase assay, wild-type or mutant EGFP-PLK3 was immunoprecipitated using GFP Trap, washed three times in IP buffer and incubated with casein in kinase buffer (10?mM HEPES pH?7.4, 5?mM MgCl2, 2?mM EGTA, 1?mM DTT, 2.5?mM -glycerolphosphate, 100?M ATP and CCG-1423 CCG-1423 CCG-1423 5? Ci 32P–ATP) for 20 min at 30 C. Proteins were separated using SDS-PAGE, and phosphorylation was visualized by autoradiography. 2.7. Cell Fractionation RPE cells were fractionated as described before [33,34]. Briefly, soluble cytosolic fraction was obtained by incubating cells in buffer A [10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 0.05% Triton X1?00 and protease inhibitor cocktail] at 4 C for 10 min and spinning down at 1500 for 2 min. Pelleted nuclei were further extracted with an equal amount of buffer B [10 mM ARF3 HEPES pH 7.9, 3 mM EDTA, 0.2 mM EGTA, 1 mM DTT] and spinning down at 2000 for 2 min yielding a soluble nuclear fraction. Insoluble chromatin was washed with buffer B and resuspended in SDS sample buffer. 2.8. Statistical Analysis Signal intensity of the bands in Western blots was measured from biological replicates ( 3) using gel analysis plug-in in ImageJ. After background subtraction, signal was normalized to the corresponding loading control and to non-treated condition. Statistical significance was evaluated using two-tailed Students T-test in Prism 5 software (GraphPad). Values < 0.05 were considered statistically significant. 3. Results 3.1. PLK3 Localizes to Plasma Membrane, Golgi and Centrosome Subcellular localization of PLK3 has been reported controversially. Whereas some studies identified PLK3 at the plasma membrane and Golgi apparatus, others observed enrichment of PLK3 in the nucleus and nucleolus [7,9,16,19,23]. Here, we screened all commercially available PLK3 antibodies and tested them in immunoblotting and immunofluorescence. Using siRNA-mediated knock down of PLK3, we have found that most of the antibodies recognized major cross-reacting bands but failed to recognize endogenous PLK3 migrating around the electrophoretic gel in close proximity (Physique 1A,B). The only antibody that in our hands recognized endogenous PLK3 was the rabbit monoclonal.