Figure 6

Direct interaction between the carboxy-terminus of LPP and the PDZ domains of Scrib. (A) GST fused to either wild-type LPP (40 amino acids of the pre-LIM region, the three LIM domains and the tail), or a similar LPP molecule with a mutated carboxy-terminus (L612A) and GST alone were expressed in E. coli, purified and analyzed by SDS-PAGE and Coomassie Blue staining. All proteins were expressed well. Protein markers are as indicated. (B) In vitro synthesized [³⁵S]-methionine-labelled full length Scrib was incubated with immobilized GST or with either one of the above-described GST fusion proteins and allowed to interact over night at 4°C. After extensive washing, bound proteins were eluted in sample buffer, separated by SDS-PAGE and visualized by autoradiography. The amount of synthesized protein loaded as a reference on the gel corresponds to 10% of the input used in each binding experiment. (C) All four PDZ domains of Scrib (amino acids 616 – 1490), either wild-type or mutated as indicated, were synthesized in vitro and [³⁵S]-methionine-labelled. these labelled proteins were incubated with immobilized GST or with GST-LPP-LT_WT and allowed to interact over night at 4°C. Bound proteins were eluted in sample buffer, separated by SDS-PAGE and visualized by autoradiography. The amount of synthesized protein loaded as a reference on the gel corresponds to 10% of the input used in each binding experiment.