human neonatal dermal fibroblasts aspm wt  (Genlantis inc)

Journal: BMC Cell Biology

Article Title: Human ASPM participates in spindle organisation, spindle orientation and cytokinesis

doi: 10.1186/1471-2121-11-85

Figure Lengend Snippet: Organisation of the ASPM protein and its cellular distribution during mitosis . A . Structure of the 3477 amino acid human ASPM protein. The region corresponding to the microtubule-binding domain of Drosophila Asp is shown in white. The calponin homology domains (aa920-1261) are in light grey; ASNP repeats are dark grey boxes (aa316-342, 366-400); 81 IQ domains (aa1267-3225) are shown as vertical stripes; the armadillo repeat-like domain (aa3294-3327) is a black box; and the C -terminal region is depicted by diagonal stripes. The location of potential nuclear localization sequences are indicated by asterisks and the location of the peptides used to raise polyclonal antibodies, by dashed arrows. B-F . Analysis of ASPM distribution following immunostaining. HeLa cells were fixed and stained with antibodies specific for ASPM (green), anti-α-tubulin (red) and with DAPI (blue) to identify nuclei. Panels B-E utilised Anti-ASPM 216-1, whilst anti-ASPM 279-3 was used in panel F. B . ASPM is predominantly nuclear in interphase cells. Scale bar = 10 μm. C . ASPM is localised to the spindle poles during metaphase. Scale bar = 5 μm. D . A globular distribution of ASPM (green) is seen around the γ-tubulin (red) immunopositive core of metaphase HeLa cell spindle poles. DNA (DAPI staining) is shown in blue (confocal image). Scale bar = 2.5 μm. E . Single 0.5 μm confocal section through the centre of a telophase HeLa cell immunostained to reveal ASPM (green) and α-tubulin (red). In addition to broad spindle pole-associated labelling (arrowhead), ASPM also localizes to the minus ends of central spindle microtubules (arrow). Scale bar = 2.5 μm. F . A late telophase fibroblast immunostained with anti-ASPM 279-3 (green), anti-α-tubulin (red) and with DAPI (blue). ASPM is predominantly localized at the midzone of the central spindle. Scale bar = 10 μm. G . Immunoblotting of cell lysates of COS7 cells (lane 1), U2OS cells (lane 2), primary fibroblasts (lane 3) and HeLa cells (lane 4) with anti-ASPM 217-2 antibody. This identifies a protein of approximately 410 kDa in each lane. A blot stained with anti-β-actin is shown as loading control.

Article Snippet: Human neonatal dermal fibroblasts ( ASPM wt ) were obtained from Genlantis (San Diego, California).

Techniques: Binding Assay, Immunostaining, Staining, Western Blot, Control

Journal: BMC Cell Biology

Article Title: Human ASPM participates in spindle organisation, spindle orientation and cytokinesis

doi: 10.1186/1471-2121-11-85

Figure Lengend Snippet: ASPM IVS25+1G > T mutation produces a novel splice variant with decreased efficiency for spindle pole localisation . A . Schematic of the ASPM protein showing the location of the ASPM IVS25+1G > T splice site mutation (filled arrows with an upper diamond). Domains of the protein are indicated as described in the legend to Figure 1. Location of epitopes of anti-ASPM antibodies are shown beneath the protein. B . Fibroblasts at metaphase immunostained using 216-1 anti-ASPM antibody (N-terminal) and the 279-3 anti-ASPM antibody, raised against a C-terminal peptide sequence. Note the reduced ASPM localization at the mitotic spindle poles of ASPM IVS25+1G > T cells. Scale bar = 10 μm. C . Chart representing integral intensity of ASPM 216-1 antibody staining at the spindle poles of ASPM wt and ASPM IVS25+1G > T fibroblast cells. Horizontal lines indicate the average integral intensity of the immunostaining. Standard error bars are present. ASPM IVS25+1G > T was at statistically significantly lower level (asterisks) at the spindle poles than ASPM wt when compared using a paired two tailed t-test. P = < 0000.1. D . Immunoblotting of fibroblast control (WT) and ASPM IVS25+1G > T (Patient) lysates with 217-2 anti-ASPM antibody and anti β-actin. Equivalent levels of stable ASPM protein are expressed by each cell culture, indicating the IVS25+1G > T mutation does not induce nonsense mediated decay. E . Sequence analysis of exon 25-exon 26 control ASPM wt and Patient ASPM IVS25+1G > T cDNA. The point of removal of the nine nucleotides due to the IVS25+1G > T mutation is indicated by an asterisk. F . IVS25+1G > T mutation removes the exon 25 splice donor site and instigates the utilisation of an in frame splice donor site nine nucleotides downstream. The resultant ASPM IVS25+1G > T protein is lacking just three amino acids (3326-3328).

Article Snippet: Human neonatal dermal fibroblasts ( ASPM wt ) were obtained from Genlantis (San Diego, California).

Techniques: Mutagenesis, Variant Assay, Sequencing, Staining, Immunostaining, Two Tailed Test, Western Blot, Control, Cell Culture