Results: 5

1.
Figure 5

Figure 5. From: Conductin/axin2 and Wnt signalling regulates centrosome cohesion.

Centrosome cohesion is regulated by Wnt signalling. (A) Quantification of split centrosomes in MEFs after treatment with sodium chloride (NaCl), lithium chloride (LiCl) or Wnt1-conditioned media for 18 h. (B) Quantification of split centrosomes in MEFs after treatment with Wnt3A-conditioned media for the indicated time points. Lower panels: western blotting for phospho-β-catenin and β-actin of lysates from the MEFs above. (C) A model for the regulation of centrosome cohesion by Wnt signalling. Error bars indicate the s.e.m. (n>300) of at least three independent experiments. MEFs, mouse embryonic fibroblasts.

Michel V Hadjihannas, et al. EMBO Rep. 2010 April;11(4):317-324.
2.
Figure 2

Figure 2. From: Conductin/axin2 and Wnt signalling regulates centrosome cohesion.

Loss or knockdown of conductin causes centrosomal splitting. (A) Anti-γ-tubulin staining using wild-type (+/+) and conductin-knockout (−/−) MEFs. The arrowheads indicate the centrosomes. Scale bar, 4 μm. (B) Quantification of split centrosomes from (A). (C) Anti-γ-tubulin staining in U2OS cells transfected with siRNAs against luciferase (siLuc) and conductin (siCond). The arrowheads indicate the centrosomes. Scale bars, 2 μm. (D) Quantification of split centrosomes from (C). GFP centrin2 was co-transfected to identify transfected cells from which percentages were calculated. Error bars indicate the s.e.m. (n>300) of at least three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; MEFs, mouse embryonic fibroblasts; GFP, green fluorescent protein; siRNAs, small interfering RNAs.

Michel V Hadjihannas, et al. EMBO Rep. 2010 April;11(4):317-324.
3.
Figure 4

Figure 4. From: Conductin/axin2 and Wnt signalling regulates centrosome cohesion.

Phosphorylation of β-catenin regulates centrosome cohesion independently from stability and transcriptional activity. (A) Quantification of U2OS cells with split centrosomes, transfected with the indicated plasmids. (B) Western blotting for transfected β-catenin constructs (exo) in U2OS cells as indicated using antibodies against total β-catenin and β-actin. Endogenous β-catenin is also indicated. (C) TOP/FOP assays of 293T cells transfected with the indicated plasmids. (D,E) Quantification of split centrosomes in U2OS cells transfected with the indicated plasmids and siRNAs. Error bars indicate the s.e.m. (n=300–600) of at least three independent experiments. GFP, green fluorescent protein; Nek2, NIMA-realted kinase 2; pm, phospho-mimicking; siRNAs, small interfering RNAs; YFP, yellow fluorescent protein.

Michel V Hadjihannas, et al. EMBO Rep. 2010 April;11(4):317-324.
4.
Figure 3

Figure 3. From: Conductin/axin2 and Wnt signalling regulates centrosome cohesion.

Conductin regulates phosphorylation of β-catenin at the centrosomes. (A) Immunofluorescence co-staining of transfected Flag-tagged conductin, Δ338–472 mutant and axin1 (red) together with γ-tubulin (green, indicated by arrowheads) in conductin-knockout (−/−) MEFs. Bottom left panel: quantification of split centrosomes. (B) Quantification of split centrosomes in U2OS cells transfected with the indicated siRNAs. The bottom panels show western blotting from these cells using the indicated antibodies. (C) Immunofluorescence co-staining for phospho-β-catenin (red) and γ-tubulin (green) in wild-type (+/+) and conductin-knockout (−/−) MEFs. The insets show a magnified view of the centrosomes. (D) Immunofluorescence staining of phospho-β-catenin (red) in U2OS cells co-transfected with the indicated siRNAs and GFP centrin2 (green). The percentage of cells showing staining for phospho-β-catenin at the centrosomes is indicated on the right. (E) Immunofluorescence co-staining of phospho-β-catenin (red) and transfected full-length conductin and deletion mutants (green) in SW480 cells. (A, B, D) Error bars indicate the s.e.m. of at least three experiments; n>200, n>400, n>300, respectively. C-Nap1, centrosomal NIMA-related kinase 2 (Nek2)-associated protein 1; DAPI, 4′,6-diamidino-2-phenylindole; MEFs, mouse embryonic fibroblasts; GFP, green fluorescent protein; siRNAs, small interfering RNAs.

Michel V Hadjihannas, et al. EMBO Rep. 2010 April;11(4):317-324.
5.
Figure 1

Figure 1. From: Conductin/axin2 and Wnt signalling regulates centrosome cohesion.

Conductin localizes at the centrosomes by binding to C-Nap1. (A) Western blotting for conductin, γ-tubulin and β-actin in sucrose density-gradient fractions from SW480 cells. The sucrose percentage is indicated. (B) Immunofluorescence co-staining of Flag conductin, GFP conductin and CFP axin2 (human conductin sequence) in green, together with γ-tubulin (red), in U2OS cells. The bottom panels show co-staining of endogenous conductin (green) with γ-tubulin (red) in SW480 cells, Scale bar, 2 μm (C) Western blotting for conductin and C-Nap1 in anti-myc immunoprecipitations using U2OS cells co-transfected with the indicated plasmids. The top panel shows the lysates (input). (D) Immunofluorescence staining of U2OS cells co-transfected with GFP C-Nap1 (green) and Flag conductin or Flag axin1 (top and bottom half, respectively; red). The insets show C-Nap1-positive centrosomes. The lower panels in the top and bottom halves show GFP C-Nap1 localization in the cytoplasmic patches. (E) Centrosomal localization and C-Nap1 colocalization of conductin mutants. (F) GFP and γ-tubulin (red) staining in U2OS cells co-transfected with GFP conductin (green) and siRNAs against luciferase (siLuc), C-Nap1 (siC-Nap1) or rootletin (siRoot). Scale bar, 2 μm (G) Co-staining of γ-tubulin and endogenous conductin in SW480 cells transfected with siLuc or siC-Nap1 (two examples shown). The coloured circles indicate the centrosomes used for quantification of the fluorescence intensities of conductin normalized to γ-tubulin intensities (shown in the bar chart). Error bars indicate the s.e.m. (n=100) of two independent experiments. C-Nap1, centrosomal NIMA-related kinase 2 (Nek2)-associated protein 1; CFP, cyan fluorescent protein; GFP, green fluorescent protein; siRNAs, small interfering RNAs.

Michel V Hadjihannas, et al. EMBO Rep. 2010 April;11(4):317-324.

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