In vitro transcription/translation and GST pull-down assays
All in vitro translation reactions were carried out using the TNT T7 Quick Coupled Transcription/Translation System (Promega) following the manufacturer's instructions. For GST pull-down assays, bacterial expression constructs were made using pGEX-5X vectors (Amersham-Pharmacia Biotech) directing the synthesis of glutathione S-transferase (GST) fusion proteins containing wild-type or mutated forms of human LPP. These fusion proteins were purified according to manufacturer's instructions and verified by SDS-PAGE. GST fusion proteins or GST alone, bound to glutathione-agarose beads, were incubated with in vitro synthesized [35S]-methionine-labelled full length human Scrib protein, or a portion of the human Scrib protein encompassing all four PDZ domains (amino acids 616–1490) (wild-type or mutated) in NENT100 buffer (100 mM NaCl, 20 mM Tris-HCl pH = 7.6, 1 mM EDTA, 0.1% NP-40, protease inhibitors). This mixture was tumbled overnight at 4°C. Subsequently the beads were washed 5 times in 500 μl NENT100 buffer, resuspended in 25 μl SDS-PAGE sample buffer and incubated at 95°C for 5 minutes. Proteins were separated by SDS-PAGE and interacting Scrib was detected by autoradiography.