Figure 2.
In vitro association of EBP50 and YAP65. (A) Lysates of 16HBE14o− cells (100 μg) were incubated with immobilized GST or GST-EBP50 (10 μg), washed, and analyzed by SDS-PAGE followed by immunoblotting with rabbit anti-YAP65 antisera. (B) Primary sequence of biotinylated peptides used in binding and competition experiments. (C) Biotinylated peptides (10 μg) were immobilized on streptavidin agarose and incubated with 16HBE14o− cell lysates (100 μg). Bound (B) and unbound (U) fractions were electrophoresed on 10% SDS-PAGE gels and analyzed by immunoblotting using rabbit anti-EBP50 antisera. (D) Radiolabeled EBP50 was generated by coupled in vitro transcription/translation in the presence of [35S]methionine and incubated with immobilized wild type (CFTRwt, YAP65wt), mutant COOH-terminal (CFTRmut, YAP65mut) or YAP65 WW domain peptides (10 μg). Bound and unbound fractions were analyzed by SDS-PAGE and phosphorimage analysis.