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. 2016 Aug 25;7:12648. doi: 10.1038/ncomms12648

Figure 1. Ubiquitin ligase RNF20/40 interacts with the motor protein Eg5.

Figure 1

(a) Immunoaffinity purification of RNF20-containing protein complexes. Cellular extracts from HeLa cells stably expressing FLAG (vector) or FLAG-RNF20 were immunopurified with anti-FLAG affinity columns and eluted with FLAG peptides. The eluates were resolved by SDS–PAGE and silver-stained. The protein bands were retrieved and analysed by mass spectrometry. (b) The RNF20/40 complex interacts with Eg5 in vivo. Immunoprecipitation assays were performed with antibodies against the indicated proteins followed by immunoblotting in HeLa, MCF-7, and PANC-1 cells. (c) Co-fractionation of Eg5 and the RNF20/40 complex by fast protein liquid chromatography. Cellular extracts from MCF-7 cells were fractionated on Superose 6 size exclusion column. The chromatographic profile with the elution positions of calibrating proteins of known molecular mass is shown. The chromatographic fractions were analysed by western blotting with antibodies against indicated proteins. (d) Eg5 interacts directly with RNF20 and RNF40 in vitro. GST pull-down assays were performed with GST-Eg5 and in vitro transcribed/translated RNF20 and RNF40. (e) Identification of the domains responsible for the direct interaction between Eg5 and RNF20 or RNF40. Bacterially purified GST, GST-Eg5N, GST-Eg5M or GST-Eg5C was incubated with in vitro transcribed/translated RNF20 or RNF40, after which GST pull-down assays were performed.