Immunoprecipitation and western blot analysis

For detection of Tyr phosphorylated adhesion proteins, or to detect changes in the interactions between α- and β-catenin, cells treated with indicated siRNAs or pervanadate (1 mM) as a positive control were lysed in 500 µl MCLB + PhosSTOP (Roche) + pervanadate (1 mM) per two 35 mm wells on ice for 20 min. Clarified lysates were normalized for total protein concentration using BCA assay (Thermo-Fisher Scientific), and 5% of the lysate was reserved for western blot analysis. An antibody directed against the endogenous β-catenin (250 µg) was added together with 30 µl of a 50% prewashed protein A/G bead slurry (Santa Cruz) and rocked for 4–12 hours at 4 °C before washing the beads and resuspended the sample in diluted sample buffer (Boston Bioproducts) and boiling in preparation for western blot analysis. Blots of the immunoprecipitated proteins were analyzed for phospho-Tyr signal or for co-precipitating α-catenin using the appropriate antibodies. Blots are representative of three independent experiments, with the exception of the preliminary experiment shown in Fig. 3B, which was carried out once as a prelude to the identification of β-catenin as the candidate protein affected by DUSP23 knockdown.

For validation of IP/MS interactions using western blot analysis, protein complexes were prepared as described above for mass spectrometry and the elutions were prepared by boiling with sample buffer (Boston Bioproducts). Blots were incubated with the appropriate antibody listed above. For IP/MS validation, two independent experiments were run on the same blot next to a control HA-GFP IP.

All western blots were visualized using IRDye 680LT Goat anti-mouse or IRDye 800CW Goat anti-rabbit secondary antibodies (Li-cor #926-68020, 926-32211) and a Li-cor Odyssey imager.