FIG 4.

MC159 coimmunoprecipitates with ubiquitin but is not covalently linked to ubiquitin. Subconfluent HEK 293T cellular monolayers were cotransfected with 500 ng pUb-Myc and 500 ng pMC159. At 24 h posttransfection, cells were incubated in medium either lacking (A) or containing (B) 10 ng/ml TNF. After 15 min, cells were lysed in Ub lysis buffer. A portion of each lysate was set aside to monitor protein expression. For the remaining clarified cellular lysates, IP was performed by using IgG or anti-Myc antibodies conjugated to protein G-Sepharose beads. Immunoprecipitated samples or cellular lysates were separated by SDS-PAGE, and proteins were transferred onto PVDF membranes for IB. Membranes were probed with the indicated antibodies to detect either MC159, Myc-tagged ubiquitinated proteins, or actin.