Anti lypla1

Immunoprecipitation was performed by cross-linking 0.25 μg/μl of antibody to 20 μl of Dyna beads (Thermo Fisher Scientific). Beads were incubated at 4°C for 90 minutes with 50 μl of egg extract. Beads were washed twice with phosphate-buffered saline (PBS) buffer and twice with PBS + 0.5 M NaCl, before they were resuspended in Laemmli sample buffer and subjected to Western blot analysis. Antibodies used for western blotting and immunoprecipitations include anti-GFP JL8 monoclonal (Clontech), anti-importin α, kif2a, and TPX2 as mentioned above, anti-XCTK2 (gift from C. Walczak, anti-xkid (gift from H. Funabiki, Rockefeller University), anti-LYPLA1 (abcam), and anti-PORCN (abcam). Immunoblotting utilized 1:1000 dilution of all antibodies or antisera except TPX2 which was used at 1:500 dilution and was performed as described (Loughlin et al., 2011 (link)). Microtubule associated proteins were isolated from Xenopus extract as described (Budde et al., 2005 ) and quantified by Western blot. Blots were scanned with an Odyssey Infarer Imaging System (LI-COR Biosciences). Alternatively, samples were processed further for detection of palmitoylation.