Proteomic analysis of INF2-associated proteins in human podocytes

GFP–INF2–WT was subcloned into pWPXL [pWPXL was a gift from Didier Trono (École polytechnique fédérale de Lausanne, Addgene plasmid # 12257)] and together with packaging vectors pMDG.2 and psAX2 [pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259) and psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260)] transfected into Lenti-X 293T Cell Line (Clontech). Human podocytes were transduced with GFP or GFP–INF2 lentivirus with 8 μg/ml polybrene overnight, the cells were thermo switched and differentiated for a minimum of 10 days [18 (link)]. 2× 175 tissue culture flasks were lysed and GFP and GFP–INF2 protein and interacting proteins were immunoprecipitated using the GFP-Trap system (Chromotek). Samples were separated on Nupage 4–12% precast gels (Invitrogen) and subjected to LC–MS/MS analysis on an Orbitrap Velos (Thermo) mass spectrometer as described previously [19 (link),20 (link)]