Nbp180586

Protein lysates from embryos were prepared by homogenising tissue in ten volumes of urea lysis buffer, as described previously (Daxinger et al., 2013 (link)). These were quantified using a BCA assay (Thermo Scientific, VIC, Australia), separated on polyacrylamide gels (Bio-Rad, Gladesville, NSW, Australia) and immunoblotted with antibodies directed against Wiz (NBP180586, Novus Biologicals, Littleton, USA) and against γ-Tubulin (T5192, Sigma Aldrich, Castle Hill, AUS). Clarity Western ECL substrate was used for visualisation (Bio-Rad, Gladesville, NSW, Australia).

Nuclear extracts were prepared from either pooled embryonic brains (E13.5) or adult cerebellum using the Nuclear Complex Co-IP Kit (54001, Active Motif, Carlsbad, CA, USA) as described previously (Harten et al., 2015 (link); Isbel et al., 2015 (link)). For each sample, 500 µg of nuclear lysate was incubated with 2 µg of anti-Wiz antibody (NBP180586, Novus Biologicals, Littleton, USA) or anti-IgG antibody (sc-2345, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 degrees Celsius. Immunoprecipitations were carried out using bead-conjugated Protein G (Dynabeads 10003D, Invitrogen, Mount Waverley, VIC, Australia) as per the Nuclear Complex Co-IP Kit manufacturer’s instructions. Eluted proteins were analysed via mass spectrometry at the La Trobe University Mass Spectrometry Facility, and the data were analysed as described previously (Harten et al., 2015 (link)).