WWOX Protein Interactome Identification

MDA-MB435S (Ev, WWOX or WFPA overexpressing) cells were seeded in 14 cm plates. Twenty-four hours later whole cell extract was prepared using lysis buffer (NaCl 150 nM, Tris 50 nM, glycerol 5%–10%, NP-40 1%, PH = 7.4) supplemented with protease and phosphatase inhibitors. A preclearing step was performed using mouse anti-IgG (Invitrogen). For IP, a cocktail of mouse anti-WWOX monoclonal antibodies^{60 (link)} was used together with protein A/G plus agarose bead (Santa Cruz, Sc-2003); they were incubated together overnight while rotating at 4 °C. Beads were washed 3 times with washing buffer (NaCl 150 nM, Tris 50 nM, glycerol 5%, NP-40 0.05%, pH = 7.4). Proteins were then eluted using two elution buffers: Elution buffer 1—2 M urea, 50 mM Tris-HCl (pH 7.5), 1 mM DTT and 5 µg/ml trypsin; Elution buffer 2—2 M urea, 50 mM Tris-HCl (pH 7.5) and 5 mM Chloroacetamide (Supplementary material^{62 (link)}). The eluted material was then injected into the MS machine (Q Exactive Mass Spectrometer, Thermo Scientific). Raw data was analyzed for putative hits in the WWOX (wild type) versus both EV (empty vector) and WFPA (mutated WWOX) groups. IP-MS was preformed using three biological replicates.