Fig. 2. In vitro interactions between FCA and FY. (A-D) The FY C-terminal PPLPP motifs interact with the FCA WW domain. (A) Autoradiograph of an acrylamide gel loaded with in-vitro translated, ³⁵S labelled FY proteins present before and after binding to GST-FCA proteins. 10% input (IN) lanes were loaded to represent FCA pre-binding. The lanes adjacent to each input lane show FY expressed as either 7xWD repeats (WD) or the C-terminal domain (CT) that was retained after binding to either GST-FCA-WW (GST-WW) or GST-FCA-WF (GST-WF). (Right) A Coomassie Blue-stained acrylamide gel showing that GST-WW and GST-WF proteins were present in equivalent amounts. (B,D) The CT domain carries two PPLPP motifs (B), which were mutated to stretches of alanines (D). (C) CT domains with either PPLPP-1 (AA-1) or PPLPP-2 (AA-2) or both (AA-1,AA-2) mutated to alanines were tested for binding to GST-WW.