Sc 166303

Protein extraction and quantitation were performed as described previously.18 (link) Proteins were taken from each sample and mixed with IgG and an appropriate amount of A/G beads (30 μL). Next, the samples were shaken gently for 4 h on ice. The mixtures were centrifuged at 1000 rpm for 5 min at 4°C, and the supernatant was transferred to a new 1.5-mL Eppendorf tube. Each sample was divided into two equal parts, which were incubated with a primary antibody against PWP1 (Santa Cruz Biotechnology, Inc., #SC-390188, 3 µg/IP), DVL2 (Santa Cruz Biotechnology, Inc., #SC-166303, 3 µg/IP), or NF2 (Santa Cruz Biotechnology, Inc., #SC-55575, 3 µg/IP), or an isotype-matched control IgG. The samples were shaken on ice for 4 h, 40 µL Sepharose A/G beads were added, and the samples were shaken on ice overnight. Each precipitate was washed three times on ice (with NP-40 protein lysate and protease inhibitors added), and each supernatant was discarded after centrifugation at 1000 rpm for 5 min at 4°C. Next, 2× loading buffer (diluted in NP-40) was added, and each sample was boiled and stored at −20°C. Electrophoresis was performed within 1 week.