Figure 5. Abnormal accumulation of... | Rockefeller University Press

Figure 5.

$Figure 5. Abnormal accumulation of katanin p60 in PP4c−/− MEF cells. (A) Overexpression of katanin p60 in MEFs resulted in defective MT array, which was seen in PP4c−/− MEF cells. Statistical analysis was performed (right; n = 50 in each group). (B) Abnormal katanin p60 accumulation at the centrosome in PP4c−/− MEF cells 48 h after infection of PP4ccko/cko MEF cells with adeno-Cre. Uninfected PP4ccko/cko MEF cells were used as controls. (top) MEF cells of each genotype were stained with an anti–katanin p60 antibody. Fluorescence intensity was calculated using ImageJ software. (bottom) Fluorescence intensity in arbitrary units (*, P < 0.05; one example of three independent experiments; n = 100). Error bars represent SEM. (A and B) Arrowheads indicate the positions of centrosomes. (C) Immunoblotting analysis of sucrose gradient fractions of centrosomal extracts using antibodies against γ-tubulin, phospho-T-219 NDEL1, and katanin p60. Proteins were extracted from uninfected PP4ccko/cko MEF cells or PP4c−/− MEF cells 48 h after infection of PP4ccko/cko MEF cells with adeno-Cre and were subjected to sucrose density gradient fractionations. One example of three independent experiments is shown. Note the presence of phosphorylated NDEL1 with γ-tubulin in PP4c−/− MEF cells and a more restricted distribution of katanin p60 in the central fractions with phosphorylated NDEL1. Bars, 10 μm.$

Abnormal accumulation of katanin p60 in PP4c^−/− MEF cells. (A) Overexpression of katanin p60 in MEFs resulted in defective MT array, which was seen in PP4c^−/− MEF cells. Statistical analysis was performed (right; n = 50 in each group). (B) Abnormal katanin p60 accumulation at the centrosome in PP4c^−/− MEF cells 48 h after infection of PP4c^cko/cko MEF cells with adeno-Cre. Uninfected PP4c^cko/cko MEF cells were used as controls. (top) MEF cells of each genotype were stained with an anti–katanin p60 antibody. Fluorescence intensity was calculated using ImageJ software. (bottom) Fluorescence intensity in arbitrary units (*, P < 0.05; one example of three independent experiments; n = 100). Error bars represent SEM. (A and B) Arrowheads indicate the positions of centrosomes. (C) Immunoblotting analysis of sucrose gradient fractions of centrosomal extracts using antibodies against γ-tubulin, phospho-T-219 NDEL1, and katanin p60. Proteins were extracted from uninfected PP4c^cko/cko MEF cells or PP4c^−/− MEF cells 48 h after infection of PP4c^cko/cko MEF cells with adeno-Cre and were subjected to sucrose density gradient fractionations. One example of three independent experiments is shown. Note the presence of phosphorylated NDEL1 with γ-tubulin in PP4c^−/− MEF cells and a more restricted distribution of katanin p60 in the central fractions with phosphorylated NDEL1. Bars, 10 μm.