Anti β catenin h 102

Pools of stage 10 Xenopus embryos were lysed in NP-40 buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris, pH 8.0, 10 µl/embryo) on ice. Samples were centrifuged for 10 mins at 13,000 rpm and the supernatant removed to a clean tube. Supernatant was then pre-incubated with protein G beads (Bio-Rad, # 1614023) for 1 hour, centrifuged to collect supernatant, and incubated with primary antibody (Anti-β-catenin H-102, Santa Cruz, sc-7199) overnight. protein G beads were then added to the sample solution and incubated for 3 hrs at 4oC. Lysate was centrifuged and the supernatant discarded. The beads were washed and centrifuged with 0.1% PBS-Tween six times. 2x SDS loading dye was added to the beads and heated to 95oC for five mins. The sample was centrifuged again and the lysate moved to a clean tube. For the active Rap1 and Rap2 pull down experiments, lysate was incubated with GST-RalGDS-RBD fusion protein overnight at 4o C to allow binding, free protein was washed away, and active Rap1 and Rap2, as well as interacting proteins, were then eluted. The active rap detection kit (Cell signaling Technologies #8818) was used as per the manufacturer’s instructions. Samples were analyzed by Western blot.