FIGURE 4.

Sh3rf2 and POSH physically interact in mammalian cells. A, Sh3rf2 co-immunoprecipitates (IP) with POSH. Myc-tagged ΔZn-POSH was co-expressed with a FLAG-tagged RING mutant of Sh3rf2 (mRING2) or empty pCMS-EGFP vector. Lysates were collected 20 h later, before significant death occurred. An aliquot of total lysate was reserved and the remaining sample was immunoprecipitated with anti-FLAG beads and the immunoprecpitates were subjected to Western immunoblotting and probed with anti-Myc. Lysates were also subjected to immunoblot for Myc to confirm equal expression of POSH under both conditions. B, mouse cortical neurons were treated with the proteasome inhibitor MG132 (20 μm) for 20 h. Cells were collected, lysed in immunoprecipitation (IP) buffer, and split into equal quantities for immunoprecipitation with anti-POSH or mouse IgG. Immunoprecipitates were resolved by SDS-PAGE and immunoblots were performed for Sh3rf2. Total cell lysate was examined in parallel. The experiment was performed three times with similar results.