Supplementary Figure 3: Characterization of Venus-Mad2 RPE1 clones and estimation of the number of molecules of Venus-Mad2 in a cell and at kinetochores.

(a) Genomic PCR screen for correctly targeted Venus-Mad2 integrants following flow cytometry selection. Primer sets consisted of an oligonucleotide annealing outside the targeting construct (either 5′ (#1) or 3′ (#4)) and an oligonucleotide annealing to the ORF of Venus (2 or 3). (b) PCR analysis with different primer combinations of a positive clone compared with parental RPE1 cells. Note that in lane 1+4 the upper band corresponds to the targeted allele whereas the bottom band corresponds to the wild-type allele. The asterisk shows a hybrid product caused by the annealing of DNA from the two other bands. (c) Localisation of Venus-Mad2 in prometaphase and metaphase. Candidate Venus-Mad2 RPE1 cells expressing ectopic CENP A-Cerulean were imaged by spinning-disk confocal microscopy. Note the co-localisation of the two signals in prometaphase that disappears in metaphase once all the kinetochores are attached. (d) Asynchronous RPE1 cells were treated similarly to Fig.3c, fixed with methanol and processed for immunofluoresence. Mad2 foci that colocalised with ACA staining were counted (n = 20 cells for nocodazole; n = 7 cells for Taxol and n = 10 cells for DMA) and compared to the data obtained by live-cell imaging (Fig. 3d,e). Error bars indicate s.d.. and are representative of at least two independent experiments. (e) Quantification of Venus-Mad2 molecules in protein extracts by immunoblotting using an anti-Mad2 antibody and recombinant His₆-Mad2. Mitotic Venus-Mad2 RPE1 cells were collected by mitotic shake-off after nocodazole treatment, counted and processed for immunoblotting. Protein extracts were prepared from known numbers of cells. Molecular mass markers on the left. (f) Calibration curve for the Mad2 antibody obtained by quantifying band intensities in (e). The dotted line represents the linear regression. The inset graph shows the frequency distribution of Venus-Mad2 fluorescence in cells treated with nocodazole and analyzed by confocal microscopy (n = 100 cells). Note that by knowing the mean number and mean fluorescence of Venus-Mad2 molecules per cell we can calculate the mean fluorescence per molecule of Venus-Mad2. (g) A maximum intensity projection of 50 z -sections taken at 0.5 μm intervals was used to identify individual kinetochores. (h) An inner circle (red) was drawn around the single kinetochore. An outer circle (blue) just around the inner circle was used to measure the local background around the kinetochore. (i) Since kinetochore signals span more than one 0.5 μm z-section, sum intensity projections were used to measure the integrated fluorescence intensity. (j) Equation used to obtain the total fluorescence intensity (Ftot) of a single kinetochore. This takes into account the local background and the respective areas of measurements. Note that all integrated fluorescence intensities were initially corrected for camera noise by subtracting fluorescence intensities of regions adjacent to cells. Scale bar = 10μm.