Ound constructs by Flag or Plk1 immunoblotting. The remainder was then

Ound constructs by Flag or Plk1 immunoblotting. The remainder was then divided and incubated in kinase buffer, with or without 200 nM BI-2536, plus 5 g GST-S, 10 M ATP, 10 mM dithiothreitol and 2.5 Ci ATP for 40 min at 30C. 32P incorporation was observed by SDS-PAGE and Typhoon TRIO imager. Three kinase assays were performed from two independent pulldowns. Signal intensity 49 quantification was performed in ImageJ by calculating area underneath the curve for each lane after background subtraction. Final construct intensities were determined by subtracting signal with BI-2536 from signal without BI-2536 and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985561 expressed as a percentage of the control. Nat Chem Biol. Author manuscript; available in PMC 2016 October 04. Lera et al. Page 12 To prepare the MedChemExpress Danoprevir Plk1-specific substrate used in the kinase assays, a single nucleic acid sequence encoding 6 known, serine-only, Plk1-specific peptide sequences identified 50 from PhosphositePlus was cloned into the pGEX-6P1 vector by Gibson assembly. Vector expression was PCI-32765 induced in E. coli by addition of 400 M IPTG for 3 hours at 37 C. Bacteria were resuspended in PBS containing 250 mM NaCl, 10mM EGTA, 10 mM EDTA, 0.1% Tween-20, 1 mM dithiothrietol, 1 mM PMSF, and 1 mg/ml lysozyme prior to sonication. GST-S was purified from lysates using Glutathione Sepharose 4B beads and eluted in 50 mM Tris-HCL pH 8.0. Immunofluorescence Microscopy General procedures–Cells were seeded on glass coverslips at low density in 24-well plates and allowed to grow until 80-90% confluence. For chromosome alignment experiments, cells were challenged for 2 h with 10 M MG-132 to prevent anaphase onset and 500 nM 3-MB-PP1 200 nM BI-2536. For chromosome segregation experiments, cells were challenged for 6 h with 200 nM 3-MB-PP1 200 nM BI-2536. For extraction experiments, cells were challenged overnight with 0.2 g/mL nocodazole. For pre-extraction, coverslips were initially incubated for 15 s at room temperature in PHEM buffer with 0.5% Triton X-100. Otherwise, coverslips were fixed in 4% paraformaldehyde in PHEM buffer for 10 min at RT, washed 3 times in PBS, and then blocked for 30 min at RT in 3% bovine serum albumin and 0.1% Triton X-100 in PBS. Primary antibodies were pooled and diluted in PBSTx+BSA. Coverslips were incubated in primary antibodies for 1 h at RT and washed 3 times in PBSTx. Alexa Fluor secondary antibodies were pooled and diluted at 1:350 in PBSTx+BSA. Coverslips were incubated in secondary antibodies for 30 min at RT and then washed twice with PBSTx. Coverslips were counterstained with DAPI and mounted on glass slides with Prolong Gold anti-fade medium and allowed to cure overnight. Image acquisition was performed on a Nikon Eclipse Ti inverted microscope equipped with a 100x/1.4NA DIC oil immersion objective, motorized stage, and CoolSNAP HQ2 CCD camera. Optical sections were taken at 0.2 m intervals and, except for extraction experiments, deconvolved using the AQI 3D Deconvolution module in Nikon Elements. Panels were cropped using Photoshop CS5 and assembled with overlays using Illustrator CS5. During quantitation of spindle polarity, chromosome alignment and segregation phenotypes, observer blinding was performed by slide label concealment. “Bipolar” spindles were defined by tubulin staining that exhibited an oval shape, tapered at opposite ends, with pericentrin signal at each end. All other spindle types were considered “abnormal”. For chromosome alignment, all non-prophase pre-anaphase mitotic cells were.Ound constructs by Flag or Plk1 immunoblotting. The remainder was then divided and incubated in kinase buffer, with or without 200 nM BI-2536, plus 5 g GST-S, 10 M ATP, 10 mM dithiothreitol and 2.5 Ci ATP for 40 min at 30C. 32P incorporation was observed by SDS-PAGE and Typhoon TRIO imager. Three kinase assays were performed from two independent pulldowns. Signal intensity 49 quantification was performed in ImageJ by calculating area underneath the curve for each lane after background subtraction. Final construct intensities were determined by subtracting signal with BI-2536 from signal without BI-2536 and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985561 expressed as a percentage of the control. Nat Chem Biol. Author manuscript; available in PMC 2016 October 04. Lera et al. Page 12 To prepare the Plk1-specific substrate used in the kinase assays, a single nucleic acid sequence encoding 6 known, serine-only, Plk1-specific peptide sequences identified 50 from PhosphositePlus was cloned into the pGEX-6P1 vector by Gibson assembly. Vector expression was induced in E. coli by addition of 400 M IPTG for 3 hours at 37 C. Bacteria were resuspended in PBS containing 250 mM NaCl, 10mM EGTA, 10 mM EDTA, 0.1% Tween-20, 1 mM dithiothrietol, 1 mM PMSF, and 1 mg/ml lysozyme prior to sonication. GST-S was purified from lysates using Glutathione Sepharose 4B beads and eluted in 50 mM Tris-HCL pH 8.0. Immunofluorescence Microscopy General procedures–Cells were seeded on glass coverslips at low density in 24-well plates and allowed to grow until 80-90% confluence. For chromosome alignment experiments, cells were challenged for 2 h with 10 M MG-132 to prevent anaphase onset and 500 nM 3-MB-PP1 200 nM BI-2536. For chromosome segregation experiments, cells were challenged for 6 h with 200 nM 3-MB-PP1 200 nM BI-2536. For extraction experiments, cells were challenged overnight with 0.2 g/mL nocodazole. For pre-extraction, coverslips were initially incubated for 15 s at room temperature in PHEM buffer with 0.5% Triton X-100. Otherwise, coverslips were fixed in 4% paraformaldehyde in PHEM buffer for 10 min at RT, washed 3 times in PBS, and then blocked for 30 min at RT in 3% bovine serum albumin and 0.1% Triton X-100 in PBS. Primary antibodies were pooled and diluted in PBSTx+BSA. Coverslips were incubated in primary antibodies for 1 h at RT and washed 3 times in PBSTx. Alexa Fluor secondary antibodies were pooled and diluted at 1:350 in PBSTx+BSA. Coverslips were incubated in secondary antibodies for 30 min at RT and then washed twice with PBSTx. Coverslips were counterstained with DAPI and mounted on glass slides with Prolong Gold anti-fade medium and allowed to cure overnight. Image acquisition was performed on a Nikon Eclipse Ti inverted microscope equipped with a 100x/1.4NA DIC oil immersion objective, motorized stage, and CoolSNAP HQ2 CCD camera. Optical sections were taken at 0.2 m intervals and, except for extraction experiments, deconvolved using the AQI 3D Deconvolution module in Nikon Elements. Panels were cropped using Photoshop CS5 and assembled with overlays using Illustrator CS5. During quantitation of spindle polarity, chromosome alignment and segregation phenotypes, observer blinding was performed by slide label concealment. “Bipolar” spindles were defined by tubulin staining that exhibited an oval shape, tapered at opposite ends, with pericentrin signal at each end. All other spindle types were considered “abnormal”. For chromosome alignment, all non-prophase pre-anaphase mitotic cells were.