Mouse anti igg

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.

To validate the WWOX-DVL2 interaction, 2 µg of 2Myc-WWOX and Flag-DVL2 were co-transfected in HEK293T cells. Twenty-four hours post-transfection, the cells were incubated with or without Wnt ligands for another 24 h. Whole cell extracts were isolated, pre-cleared with mouse anti-IgG (Invitrogen) and were subjected to IP overnight using anti-Myc (Santa Cruz, Sc-40), anti-IgG and anti-Flag (Sigma Aldrich, F1804) antibodies. Precipitates were washed, eluted and run on SDS-PAGE for immunoblotting with the indicated antibodies.