Supplementary Figure 1: Initial characterization of Kif25.
(a) RT-PCR was carried out as described in Methods to show endogenous Kif25 expression in HeLa cells. Kif18A was used as a positive control and showed signal above threshold (Ct) at cycle 15, Kif25 signal increased over threshold at cycle 20 consistent with its expression in our HeLa cells. Inset shows emission of SYBR green/emission of ROX dye, Rn, as a measure of amplification. Negative controls in which the cDNA or the reverse transcriptase were omitted from the reaction showed no significant amplification of signal, the no cDNA reaction shown passed threshold at cycle 36 consistent with no significantly amplified message, error bars represent mean ± S.D. for n = 3 experiments. (b) Cells were treated for 72 hr on 100 cm dishes with either control siRNA or one of two separate Kif25 siRNA constructs. Cell lysates were separated by SDS-Page. Proteins transferred to nitrocellulose membranes were stained with polyclonal anti-Kif25 antibodies (Santa Cruz Biotechnology) to show endogenous Kif25 KD. α-tubulin was used as a loading control. The experiment was performed twice. (c) Hydrodynamic analysis of purified GFP-Kif25 shows it to be a tetrameric motor. Optical scans of gel lanes from gel filtration and velocity sedimentation were quantified with ImageJ. Gaussian peaks were fit to protein gel bands and standard curves were constructed with Prism from published data for standard proteins. Three gel filtration columns, internally controlled, were run on GFP-Kif25 and two dimeric deletion mutants. Gaussian peaks were fit to data of optical scans of Coomassie-stained gel fractions. Quality of fits to the data were assessed by R-squared analysis. For gel filtration chromatograph, the mean R-squared was 0.98 +/− 0.012 (s.d., n = 12). For velocity sedimentation, the mean R-squared was 0.92 +/− 0.122 (s.d., n = 20). [Please provide information on reproducibility].
