Rabbit α gfp

Mouse αRan was from BD Transduction Laboratories (1:2,000, catalogue number: 610341); mouse αHA.11 (1:500, catalogue number: MMS-101P) was from Sigma; rabbit αGFP (1:1,000, catalogue number: sc-8334), rabbit αUbc9 (1:1,000, catalogue number: sc-10759), goat αCrm1 (1:1,000, catalogue number: sc-7826; used for immunoprecipitation experiments) and goat αSPN1 (1:1,000, catalogue number: sc-55301) were from Santa Cruz. Affinity purified goat αRanBP2 (1:1,000) and goat αRanGAP1 (1:1,000) have been described elsewhere16 (link)29 (link). Goat αCrm1* (1:1,000 from a 0.2 mg ml⁻¹ stock) was affinity purified from goat αCrm1 serum (raised against the C-terminally His-tagged, full-length Crm1 protein—see above). Uncropped western blots are shown in Supplementary Fig. 4.

Flies were raised at 25^oC and dissected when they reached the 3rd larval instar, unless otherwise stated. Dissection was performed in pre-chilled PBS and carcasses were then immediately fixed in 4% paraformaldehyde (PFA) for 20 mins, washed 3 times with PBT (PBS with 0.1% Triton X-100), and blocked with PBT with 0.1% BSA for 1 h. Samples were incubated in primary antibody overnight at 4 °C then washed 3 times with PBT and incubated in secondary antibody at room temperature (RT) for 1.5 h. Samples were then washed 3–4 times with PBT and equilibrated in PBS with 70% glycerol overnight before mounting. Brains were mounted in mounting media (Citiflour AF1) for imaging. The following primary antibodies were used: rabbit α-GFP (1:10,000, Sigma), rabbit α-Grh (1:2000, gift of Christos Samakovlis), and rabbit α-Asense (1:2000, courtesy of Y.N.Jan) [22 ], mouse α-Pros (1:100, Developmental Studies Hybridoma Bank (DSHB)), mouse α-repo (1:500, DSHB), and mouse α-Mira (1:100) [23 ], guinea-pig α-Dpn (1:5000, gift form C. Delidakis [24 ];) and α-Path (1:50, [15 ]), rat α-Elav (1:20 DHSB) Secondary antibodies for mouse, rabbit, guinea pig or rat were conjugated to Alexa 488, 555, 568, 633 or 647 (Molecular Probes) or to FITC, Cy3 or Cy5 (Jackson ImmunoResearch).