Figure 5.

TIP-1 competes with mLin-7 for binding to Kir 2.3. (A) Diagram illustrating the procedure for competitive immunoprecipitation experiments as described below and in Materials and Methods. (B) Kir 2.3-mLin-7 complexes were recovered on protein G-Sepharose beads with anti-VSV antibodies from COS cells expressing VSV-tagged Kir 2.3 and HA-tagged mLin-7. Three separate competition reactions were run in parallel. Lysate from COS cells transfected with pcDNA 3.1 was added (pc DNA lysate++) to one sample as a control. Another sample contained an equal volume of COS cell lysate containing TIP-1 (TIP-1-myc++). The other sample contained an equal volume of a 1:1 mix of the TIP-1-myc and pcDNA lysates (TIP-1-myc+/pcDNA+). After incubation at RT, HA-mLin-7, and TIP-1-myc bound to Kir 2.3 was recovered and detected by immunoblot. (C) Amount mLin-7 bound to Kir 2.3 relative to control (pC DNA++) was evaluated by densitometry of experiments shown in B. Mean ± SE; n = 3; p < 0.005.