In vitro translation

In vitro translation was performed as previously described (Merte et al., 2010 ), with slight modifications. In detail, 1 mM CaCl₂ and 10 μg/ml micrococcal nuclease were added to 100 μl of permeabilized cells prepared as described to remove endogenous RNA. After incubation at room temperature for 12 min, 4 mM ethylene glycol tetraacetic acid (EGTA) was then added to terminate the reaction. Membranes were then centrifuged at 10,000 × g at 4°C for 15 s and resuspended in KHM buffer such that the optical density at 600 nm of 5-μl membranes in 500 μl of KHM buffer was 0.060. These membranes were added to in vitro translation reactions containing rabbit reticulocyte lysate (Flexi; Promega, Madison, WI), amino acids, KCl, and mRNA encoding HA-Vangl2, which was incubated at 30°C for 60 min (Merte et al., 2010 ). Donor membranes were centrifuged at 2700 × g at 4°C for 3 min and resuspended with 1 ml of B88-LiCl buffer. Donor membranes were then washed twice with B88 buffer and resuspended in 20 μl of B88 buffer per reaction.