FIGURE 5.

CIP75 interacts with non-ubiquitinated Cx43. Sequential immunoprecipitation of ubiquitinated protein and Cx43HKKSL that interacts with CIP75 in lactacystin-treated HeLa-Cx43HKKSL cells. CIP75 was first immunoprecipitated from cellular lysates. Immune complexes were subsequently disrupted using RIPA buffer and subjected to a second immunoprecipitation of Cx43 or HA-tagged ubiquitinated proteins to isolate the subset of proteins that interact with CIP75. CIP75 interacts with Cx43HKKSL and HA-ubiquitin-tagged proteins (top and middle, lane 1). Cx43HKKSL that was initially co-immunoprecipitated with CIP75 is not ubiquitinated (top and middle, lanes 2 and 3), whereas ubiquitinated proteins that were initially co-immunoprecipitated with CIP75 are separately recovered (top, lane 3). CIP75 is not recovered after the sequential immunoprecipitation (bottom, lanes 2 and 3). *, IgG heavy chain; **, light chain. Migration positions of the molecular mass markers in kilodaltons are indicated at the right of the co-immunoprecipitation panels. Protein inputs are shown in the right panels and represent 2% of the amount of lysates used for immunoprecipitations. IP, immunoprecipitation; IB, immunoblot.