TI - Figures and Tables . AB - Figure 1 Preparation of chromosome scaffold fractions from colcemid-treated HeLa S3 cells . Proteins were separated by 12.5% ( A ) and 7.5% ( B ) SDS-PAGE and gels stained with Coomassie Blue . Apparent molecular masses are shown in kDa on the left. ( C ) Immunoblot characterisation of the partially-extracted chromosome scaffold fraction . After SDS-PAGE , proteins were transferred to a nitrocellulose membrane and strips of the same membrane were hybridised with antibodies as follows : lane 1 , anti-INCENP ; lane 2 , anti-centromere antibodies ( ACA ) ; lane 3 , anti-CENP-C ; lane 4 , anti-Aurora B ; lane 5 , anti-ScII ; lane 6 , MPM-2 monoclonal . Arrowheads indicate the CENP-C polypeptide recognised by the ACA and the CENP-C-specific antibody . Apparent molecular masses are shown in kDa on the left. ( D ) Comparison of scaffold fractions resulting from extraction under different conditions . A 12.5% polyacrylamide gel was silver stained after separation of proteins by electrophoresis . To control for protein loading , different cell equivalents were used as follows : lane 1 , total chromosomes from 106 cells ; lane 2 , partially-extracted chromosome scaffold prepared as described in this paper from 5 x 106 cells ; lane 3 , chromosome scaffolds extracted by dextran sulphate-heparin as described ( 36 ) from 12.5 x 106 cells ; lane 4 , chromosome scaffolds extracted by NaCl as described ( 36 ) from 12.5 x 106 cells . Arrowheads indicate the two abundant protein bands typically found in chromosome scaffolds , ScI and ScII . Apparent molecular masses are shown in kDa on the left . Figure 2 Putative subcellular distribution of 62 proteins identified by MALDI-TOF in the extracted chromosome fraction . Only one localisation was given for any one protein . Table 1 lists these proteins and their individual assignations to a subcellular localisation . Figure 3 Localisation of NGB/CRFG as a chromosomal protein in mitosis. ( A ) 105 HeLa JW cells were transiently transfected with 2 ug pEGFP-C1-CRFG . Cells were fixed 24 h after transfection as described , and were immunostained for alpha tubulin and stained with DAPI . Cells in interphase and in different stages of mitosis are shown . Black-and-white images show GFP-CRFG staining , which is shown in green in the merged images . Tubulin staining is shown in red and DNA in blue . Images are of single focal planes . Scale bar is 10 um. ( B ) Deconvolved image of a transfected cell in metaphase to show the perichromosomal localisation of the GFP-CRFG signal more clearly . Separate channels are shown as indicated and the merged image uses the same colour scheme as in (A) . Figure 4 ( A ) Assay for kinase activity in chromosome scaffold fraction . Lanes 1-4 show autoradiographic image of an SDS-polyacrylamide gel used to separate proteins after chromosomes in kinase buffers 1-4 , respectively , were incubated with [gamma-32P] dATP and then extracted . Apparent molecular masses are shown in kDa on the left. ( B ) Assay for Aurora B kinase SUBstrates in chromosome scaffold fraction . Chromosomes were heat-treated and then incubated in the presence of [gamma-32P] dATP without ( lanes 1 and 3 ) or with ( lanes 2 and 4 ) recombinant Aurora B and extracted before being subjected to SDS-PAGE . Lanes 1 and 2 show an autoradiographic image of a 12.5% gel and lanes 3 and 4 show the same gel stained with Coomassie Blue . Arrowheads indicate PHOSphorylated proteins and apparent molecular masses are shown in kDa on the left . Figure 5 Confirmation of topo IIalpha as an Aurora B SUBstrate in vitro , showing an autoradiograph of an SDS-polyacrylamide gel on which recombinant , heat-treated topo IIalpha was resolved following incubation with wild-type GST-Aurora B ( lanes 1-4 ) or GST-Aurora B K106R ( lanes 5-8 ) in kinase buffer 1 ( lanes 1 and 5 ) , kinase buffer 2 ( lanes 2 and 6 ) , kinase buffer 3 ( lanes 3 and 7 ) or kinase buffer 4 ( lanes 4 and 8 ) . Apparent molecular masses are shown in kDa on the left . Figure 6 Immunofluorescence localisation of topo IIalpha and Aurora B . Colcemid-treated , hypotonically swollen HeLa S3 cells were spread and immunostained as described . Deconvolved images show staining for topoisomerase II ( green ) and Aurora B ( red ) . DNA is shown in blue . Scale bar is 5 um . Table 1 .