Journal: Journal of Cell Science
Article Title: Enhancement of CEP215 dynamics for spindle pole assembly during mitosis
doi: 10.1242/jcs.263542
Figure Lengend Snippet: Generation of CEP215-truncated mutants with defects in molecular dynamicity. (A) Schematic diagram illustrating the CEP215 protein with functional sites (CM1 and CM2), protein-binding sites, coiled-coil domains (CCDs), intrinsically disordered regions (IDRs) and the truncated mutant sites used in this study. (B) The number of cells with (w/) cytoplasmic clusters of FLAG–CEP215–CRY2-truncated mutant proteins was counted. * P <0.05 (one-way ANOVA with Tukey's post hoc test). (C) FRAP signals of the mCh–CEP215–CRY2 mutant proteins at the cytoplasm were measured for up to 600 s. w/o, without. (D) Stably expressed FLAG–mCh–CEP215 mutants in p53 KO ; CEP215 KO HeLa cells were subjected to immunoblot analyses with anti-CEP215, FLAG and GAPDH antibodies. Blot representative of four experimental repeats. (E) FRAP analyses of the FLAG–mCh–CEP215 proteins were carried out at mitotic phase centrosomes of the rescued cells. GFP–centrin2 marks the centrosomes. The analyzed cells were photographed with differential interference contrast microscopy. Scale bars: 1 μm (FRAP); 10 μm (DIC). (F) The FRAP signals of FLAG–mCh–CEP215 (WT, ΔCCD5 and L1867S) at mitotic phase centrosomes were measured for up to 160 s. For B, C and F, at least 90 (B), 30 (C) and 10 (F) cells per experimental group were counted in three independent experiments. Values are mean±s.e.m. C and F show arbitrary units.
Article Snippet: For immunostaining analyses, we used rabbit antibodies against CEP215/CDK5RAP2 (06-1398; Merck Millipore; 1:100), PCNT , CEP192 (A302-324A; Bethyl Laboratory; 1:100), α-tubulin (ab18251; Abcam), γ-tubulin (ab11317; Abcam; 1:100); mouse antibodies against centrin2 (04-1624; Merck Millipore), α-tubulin (T6199; Sigma; 1:100), FLAG (F3165; Sigma; 1:100); and goat anti-FLAG (ab1257; Abcam; 1:100) antibodies.
Techniques: Functional Assay, Protein Binding, Mutagenesis, Stable Transfection, Western Blot, Microscopy
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