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information on fly lines , imaging techniques , image analysis and microinjection can be found at hayward et al . , 2014 .
the gst - rant24n construct was a gift from a. wilde and was produced as described previously .
full - length d - tpx2 ( mei-38 ) and d - hurp ( mars ) cdnas were amplified by pcr and cloned into pdest17a or pmal - c2x / dest ( a gift from jason carlyon ) vectors respectively , using the pentr / d / topo system .
constructs were transformed into bl21 competent cells with induction and purification of his - tagged and mbp - tagged proteins performed as described previously .
the microtubule co - sedimentation assay was adapted for use with recombinant proteins from hughes et al . ; using 31.25 g tubulin , 1 m gtp , pure protein ( mbp at 2 mg / ml , his - d - tpx2 at 2 mg / ml or mbp - d - hurp at 10 mg / ml ) and 25 m taxol where appropriate . the microtubule stability assay was performed using 50 l c buffer containing 62.5 g tubulin , pure protein ( same amounts as co - sedimentation assay ) and 1 mm gtp , incubated at 37 c for 25 min .
samples for both the microtubule binding and microtubule stability assays were run on standard sds - page gels and stained with coomassie .
briefly , clarified ( high speed supernatant ) embryo extracts , made from 0.4 g of 03 h old gfp - d - tpx2 or gfp - d - hurp embryos as previously described , were incubated with 30 l of gfp - trap - a bead slurry ( chromotek , germany ) in c buffer for 2 h at 4 c with rotation .
beads were washed 5 x in 1 ml c buffer , frozen in n2 ( l ) and processed by the bristol proteomics facility using orbitrap nano - lc msms .
identified proteins were cross - referenced against an in - house database of non - specific ( false - positive ) proteins identified from 3 control experiments from embryos expressing different gfp - fusion proteins in which the bait protein was not precipitated ( i.e. , negative controls ) .
table 1 constitutes proteins from the gfp - d - hurp ap - ms which : ( i ) either were not identified in negative controls or were identified in negative controls with scores of at least 4-fold less than in gfp - d - hurp and ( ii ) had an ms score of > 30 and ( iii ) had > 10% peptide : protein coverage .
the remaining augmin subunits were present specifically in gfp - d - hurp ap - ms but with scores of < 30 .
upon entry into mitosis , many microtubules are nucleated that coordinately integrate into a stable , yet dynamic , mitotic spindle apparatus . 
 in a recent publication , we examined microtubule - generating pathways within a single model system , the drosophila syncytial embryo . 
 we found that , following depolymerisation of metaphase spindle microtubules by cold treatment , spindles regenerate predominantly from microtubules nucleated within the vicinity of chromatin . 
 we also showed this chromatin - mediated microtubule nucleation is mediated by the drosophila homolog of a vertebrate spindle assembly factor ( saf ) , hurp and is dependent on the conserved microtubule amplifying protein complex , augmin . here , we expand our investigation into drosophila safs , providing evidence that , in vitro , both d - hurp and d - tpx2 are able to bind to and stabilize microtubules . we show that gfp - d - hurp purified from embryos interacts with importin- and augmin and , consistent with this , demonstrate that the underlying basis of chromatin - mediated microtubule nucleation in drosophila syncytial embryos is dependent on ran - gtp .