Journal:

Article Title: C-terminal-binding protein corepresses epithelial and proapoptotic gene expression programs

doi: 10.1073/pnas.0830998100

Figure Lengend Snippet: Epithelial phenotype-related proteins are regulated by CtBP. Equal amounts of total cellular protein from CtBP1,2−/− cells (−/−), cells rescued with vector alone (vec−/−), cells rescued with wild-type CtBP1 [res.(wt)] or the H315Q mutant [res.(H315Q)], or heterozygous control cells (−/+) were assayed for expression of desmoglein-2 (DSG-2), plakoglobin (PG), keratin-8 (K8), E-cadherin (E-cad), or occludin (Occl) by Western blotting. Confirmation of mouse CtBP (left) or human CtBP (right) expression in the mixed populations of CtBP-rescued cells and an actin-loading control is also shown; CtBP expression in >95% of the rescued cells was additionally verified by immunofluorescence (data not shown). (Lower) Epithelial genes indicated by the microarray analysis to be both down-regulated in the CtBP1,2−/+ and the CtBP-rescued cells, relative to the CtBP1,2−/− cells, are shown. The complete microarray results are published as supporting information on the PNAS web site.

Article Snippet: His-tagged CtBP and H315Q mutant were expressed in BL21 (DE3) and purified by Ni-NTA affinity (Qiagen).

Techniques: Plasmid Preparation, Mutagenesis, Control, Expressing, Western Blot, Immunofluorescence, Microarray

Journal:

Article Title: C-terminal-binding protein corepresses epithelial and proapoptotic gene expression programs

doi: 10.1073/pnas.0830998100

Figure Lengend Snippet: Dehydrogenase activity of CtBP is not required for transcriptional repression. (a) The PERP or β-actin control promoters linked to luciferase (1.5 μg) were cotransfected with the indicated CtBP mutants, and activity of the luciferase reporters was assayed. (b) GST-E1a fusion proteins coupled to glutathione-Sepharose beads were incubated with His-tagged human CtBP or H315Q mutant. The bound material was dissolved in sample buffer and analyzed by Western blotting by using an antibody against the His-tag. Equal amounts of GST and GST-E1a proteins were used in each pull-down assay, as shown (Lower). (c) An SV40 promoter adjacent to four gal4-binding sites (1.5 μg) was assayed for repression by the gal4-CtBP fusion proteins (0.5 μg), as indicated.

Article Snippet: His-tagged CtBP and H315Q mutant were expressed in BL21 (DE3) and purified by Ni-NTA affinity (Qiagen).

Techniques: Activity Assay, Control, Luciferase, Incubation, Mutagenesis, Western Blot, Pull Down Assay, Binding Assay

Journal: Nature Communications

Article Title: The Bub1–Plk1 kinase complex promotes spindle checkpoint signalling through Cdc20 phosphorylation

doi: 10.1038/ncomms10818

Figure Lengend Snippet: ( a , b ) Anti-Myc blot of the in-vitro ubiquitination reactions of APC/C Cdc20 using cyclin B1 1–97 –Myc as the substrate and UbcH10 ( a ) or both UbcH10 and Ube2S ( b ) as ubiquitin-conjugating enzymes. Recombinant Cdc20 WT or S92E at different concentrations (16.6, 66.4 and 332 nM) were incubated with APC/C isolated from Xenopus egg extract. ( c ) Quantification of the mitotic index of HeLa Tet-On parental cells and cells stably expressing Flag-Cdc20 WT or S92E that were treated with or without Cdc20 siRNA or Dox (mean±s.d.; n =3 independent experiments). ( d ) Quantification of the mitotic index of HeLa Tet-On cells stably expressing Flag-Cdc20 WT or S92E that were treated with Cdc20 siRNA, Dox and 200 nM taxol in the presence or absence of Bub1 siRNA or BI 2536 (BI) (mean±s.d.; n =3 independent experiments). ( e ) HeLa Tet-On parental cells and cells stably expressing Flag-Cdc20 WT or S92E were treated with the indicated siRNAs and 500 nM nocodazole. Cell lysates were blotted with the indicated antibodies. ( f ) Quantification of the mitotic index of cells in e (mean±s.d.; n =3 independent experiments).

Article Snippet: For protein-binding assays, purified His 6 –Cdc20 ΔN60 WT or the S92E mutant were immobilized on Ni 2+ -NTA beads (QIAGEN).

Techniques: In Vitro, Recombinant, Incubation, Isolation, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: The Bub1–Plk1 kinase complex promotes spindle checkpoint signalling through Cdc20 phosphorylation

doi: 10.1038/ncomms10818

Figure Lengend Snippet: ( a ) HeLa Tet-On cells stably expressing Flag-Cdc20 WT or 5A were arrested in mitosis by 5 μM nocodazole. The cell lysates and the anti-Flag immunoprecipitates (IP) of these cells were blotted with the indicated antibodies. The graphs at the bottom show the quantification of the relative BubR1 and Mad2 signals normalized to that of total Cdc20 in the IP (mean±range; n =2 independent experiments). ( b ) Blots of the input and beads-bound proteins of the binding reactions among the indicated proteins. The graphs at the bottom show the quantification of the relative BubR1 and Mad2 signals normalized to Cdc20. ( c ) Schematic drawing of the experimental design in d and . APC/C is pre-activated with Cdc20 WT or S92E and then incubated with mini-MCC comprising BubR1N, Mad2 in the closed conformation and Cdc20 (WT or S92E). ( d ) Anti-Myc blot of the in-vitro ubiquitination reactions of the indicated APC/C Cdc20 incubated with the indicated proteins and using securin-Myc as the substrate. The asterisk indicates a nonspecific cross-reacting band.

Article Snippet: For protein-binding assays, purified His 6 –Cdc20 ΔN60 WT or the S92E mutant were immobilized on Ni 2+ -NTA beads (QIAGEN).

Techniques: Stable Transfection, Expressing, Binding Assay, Incubation, In Vitro