(a) WWOX interacts with specific PPXY motifs of candidate interacting proteins. Site-directed mutagenesis was used to change the tyrosine of PPXY to alanine of SIMPLE (lanes 5–8) and WBP-1 (lanes 2–4). Additionally, a double mutant of SIMPLE was created (lane 8). To determine protein interactions, total protein extracts from bacteria expressing the wild-type and mutant proteins were used for GST pull-down assays. Proteins pulled down by GST–WWOX-1 and -2 were separated by SDS–PAGE, transferred to PVDF membranes and visualized by Western blotting using antibody to the histidine tag (top panel). Equal amounts of expressed proteins were determined by Western blotting of total protein extracts (bottom panel). Total protein extracts from the parental bacterial strain that does not express any histidine-tagged proteins were used as the negative control (lane 1). *Band due to nonspecific binding of the antibody to the GST protein. (b) The first WW domain is required for interaction with endogenously expressed SIMPLE. Whole-cell extracts from the breast cancer cell line MCF-7 were used for GST pull-down assays using GST fused to each individual WWOX WW domain and to GST–WWOX-1 and -2 containing altered amino acids in each of the WW domains. Proteins pulled down by the indicated GST-fusion proteins were separated by SDS–PAGE, transferred to PVDF and analysed by Western blotting with antibody that specifically recognizes SIMPLE. Left panel: Binding to the complete WWOX WW domain (GST–WWOX-1 and -2, lane 2) and to the individual WWOX WW domains (GST–WWOX-1 and GST–WWOX-2, lanes 3–4, respectively). The observed binding by endogenous SIMPLE was specific for WWOX, as shown by the GST pull-down with GST alone (lane 1). Right panel: Binding to the complete WW domain (GST–WWOX-1 and -2, lane 5) and to the mutant WW domains (mut1 and mut2, lanes 6 and 7, respectively). Each WW domain had two amino acids changed in the context of GST–WWOX-1 and -2 ()