Figure 8.

SMURF2 binding to RNF11 or SMAD7 is mutually exclusive. A, RNF11 competes with SMAD7 for SMURF2 binding. Cell lysates from insect cells expressing FLAG-SMURF2 or HA-SMAD7 were first combined (1 mg of total protein each) and incubated for 1 h to preassemble complex. Three different amounts of bacterial lysates expressing GST, GST-RNF11 WT, or GST-RNF11 Y40A were then added (0.1, 1, and 5 mg of protein) to the reaction and incubated and supplemented with glutathione-agarose for another 2 h and resolved by SDS-PAGE. Titration of GST-RNF11 WT results in loss of SMAD7 interaction with SMURF2. B, RNF11 and SMAD7 form mutually exclusive complexes with SMURF2. Cell lysates from insect cells expressing FLAG-SMURF2 or HA-SMAD7 (1 mg of total protein each) were mixed with bacterial lysates expressing GST, GST-RNF11 WT, or GST-RNF11 Y40A (1 mg of total protein each) and incubated for 2 h, and the resulting complexes were immunoprecipitated with anti-FLAG-agarose. Elution from the FLAG immunoprecipitates was performed with FLAG peptide, and 5% of the eluate was resolved by Western blotting (top). The remainder of the eluate was then immunoprecipitated a second time with glutathione-agarose (middle) and anti-HA agarose (bottom).