Searching for new members of the SLX4 complex and for SLX4-complex members that are SUMOylated in vitro

The project was initially aimed at identifying new members of the SLX4 complex as well as members of the complex that are SUMOylated in vitro. The project was also aimed at assessing whether SUMOylation may change the compositon of the complex by reducing complex association of SUMOylated partners. YFP-SLX4 complexes were immunopurified from Hela Flp-In TRex cells producing full length YFP-SLX4 using a GFP nanobody. The YFP-SLX4 pull down was used in an ex vivo/in vitro SUMO-ligation assay as described in Guervilly et al. 2015. After the SUMO-ligation reaction, beads were washed 5 times with 50 mM Tris-HCl [pH 8.0] buffer, allowing for the removal of SUMOylated proteins that associate less efficiently with SLX4, or other members of the complex. SLX4 and proteins remaining associated with SLX4 after the successive washes were eluted directly in NuPAGE LDS sample buffer (Invitrogen) for 5 min at 95˚C.