hec1 antibody epitope mapping  (Biosynth Carbosynth)

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 1. Epitope Mapping and Cellular Localization of Hec1 Monoclonal Antibody 9G3 (A) Peptides covering the sequence of human Hec1 were adsorbed onto nitrocellulose and immunoprobed with 9G3. As a control, HeLa extract was adsorbed onto the nitrocellulose at region H-12. Both the control spot and spot C-2 (amino acids 200–215) were positively identified. (B) Representation of the Ndc80 complex as predicted from previous publications (Wei et al., 2005; Ciferri et al., 2005). The asterisk marks the site on Hec1 where 9G3 binds. (C and D) Localization of 9G3 (green) and an antibody to Spc24 (red) in PtK1 cells (C) and HeLa cells (D). Linescans were carried out on sister kinet- ochore pairs from both HeLa cells (n = 40 pairs/3 cells) and PtK1 cells (n = 34 pairs/4 cells), and in all cases Hec1 localized exteriorly to Spc24 at kinetochores. (E) Western blot of whole-cell PtK1 extract with 9G3 as a probe. (F) Immunofluorescent image of a PtK1 cell injected with 9G3. To the right of each cell panel in (C), (D), and (F), a single kinetochore pair has been enlarged. The graphs represent the linescan data from the single kinetochore pair. Scale bars in (C), (D), and (F) = 5 mm.

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: Sequencing, Control, Western Blot, Injection

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 4. Loss of Kinetochore Oscillations and Plus-End MT Polymerization in Hec1 9G3-Injected Cells (A and B) Kinetochore behavior was analyzed by live-cell fluorescence timelapse imaging. Cells were injected with rhodamine-labeled tubulin and Alexa 488-conjugated CENP-F antibodies (A), or additionally with 9G3 (B). Images were acquired every 15 s. Selected planes are shown from the timelapse sequences (A and B, top). Selected kinetochore pairs are boxed and a time series of 12 images for each pair is shown below (A and B, bottom). Kinetochores from the control cell exhibited oscillatory behavior and periods of stretching and relaxation (example in A, bottom), whereas kinetochores from the 9G3-injected cell did not oscillate (example in B, bottom). (C and D) EB1-GFP-expressing PtK1 cells were injected with Texas Red dextran alone (C) or in combination with 9G3 (D). Images were acquired every 10 s. A region containing a kinetochore pair and the spindle poles was extracted from the timelapse sequence and shown to the right. The bright spots in extracted images are spindle poles. (E) A buffer-injected monopolar cell exhibits chromosome oscillations toward and away from the pole both prior to and after injection (top panel). In cells injected with 9G3, chromosomes stopped oscillating and moved poleward after injection (middle and bottom panels). In all panels, scale bars = 5 mm.

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: Injection, Imaging, Labeling, Control, Expressing, Sequencing

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 6. Aurora B Kinase Phosphoryla- tion and Regulation of Hec1 (A) Left: Aurora B/INCENP790–856 in vitro kinase assay with Histone H3 as a control substrate (lanes 1 and 2) and Hec11–230 (lanes 3 and 4). Antibody 9G3 was added to the reaction mixtures in lanes 2 and 4. Right: Normalized quantification of radioactive phosphate for lanes 1–4. Molecular weight standards are indi- cated in kilodaltons. (B) PtK1 cells were transfected with WT-GFP- Hec1 (upper row) or mutant 6A-GFP-Hec1 (bot- tom three rows) for 40 hr prior to fixation for im- munofluoresence. Scale bar = 5 mm. (C) Cells transfected with WT-GFP-Hec1 and 6A-GFP-Hec1 were scored for chromosome alignment and assigned to one of three cate- goryies: chromosomes all aligned, chromo- somes mostly aligned (1–2 chromosomes off the metaphase plate), or chromosomes mostly unaligned (fewer than 3 aligned chromosomes) (n = 52 WT-GFP-Hec1 cells; n = 40 6A-GFP- Hec1 cells). (D) Cells were scored for merotelic kineto- chores. For WT-GFP-Hec1-expressing cells: n = 19 prometaphase cells, n = 31 metaphase/ near metaphase cells, and n = 11 anaphase cells. For 6A-GFP-Hec1-expressing cells: n = 47 prometaphase cells, n = 13 metaphase/ near metaphase cells, and n = 18 anaphase cells.

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: In Vitro, Kinase Assay, Control, Molecular Weight, Transfection, Mutagenesis, Expressing

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 7. Model for Hec1 Regulation of kMT Dynamics and Attachment (A) Mitotic spindle arrangement in a control cell (top) in which normal Aurora B phophorylation and dephosphorylation occur. kMT plus ends exhibit dynamic instability and undergo periods of attachment and detachment. Net polymerization at plus ends of kMTs is balanced by net depolymerization at minus ends. After addition of 9G3 (bottom), the N terminus of Hec1 can no longer be phosphorylated by Aurora B, and kMT detachment and dy- namic instability are suppressed. Minus-end depolymerization is not inhibited, and active depolymerases shorten kinetochore fibers. Hyper-stretch of centromeres arises from pulling forces exerted by the centrosome-associated minus-end organizing complexes as they maintain connection with the depolymerizing minus ends of the kinetochore fibers. (B) Regulation of kMT plus-end dynamic instability and attachment strength at three possible interfaces. Interface 1 is between the N terminus of Hec1 and the MT lattice; interface 2 is between the N terminus of Hec1 and a kinetochore-binding MAP, and interface 3 is between the MAP and the MT lattice (see text for details).

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: Control, De-Phosphorylation Assay, Binding Assay