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. 2005 Apr;25(8):3117–3126. doi: 10.1128/MCB.25.8.3117-3126.2005

FIG. 9.

FIG. 9.

Overexpression of TCTP blocks Mcl-1 from undergoing ubiquitination. (A) CHOP cells were transiently transfected with the HA-mMcl-1 expression vector along with increasing amounts of the vector encoding FLAG-TCTP. At 16 h after transfection, cells were treated with MG-132 (2 μM, lanes 1 to 4) or dimethyl sulfoxide (vehicle control) (lanes 5 to 8) for 4 h before cell lysates were prepared and immunoprecipitated with anti-mMcl-1 antibody. The immune complex was then analyzed by immunoblotting with anti-ubiquitin (UB) antibody (top panel). The ubiquitinated form of mMcl-1 is as indicated (UB-Mcl-1). Direct immunoblotting analysis of the corresponding cell lysates (1/20 of those used in the top panel) with anti-HA and anti-FLAG antibodies are shown on the middle and the bottom panels, respectively. (B) The K257V mutant is more susceptible to undergoing ubiquitination. CHOP cells transiently overexpressing the HA-tagged wild-type (lanes 1 and 3) or K257V mutant (lanes 2 and 4) forms of mMcl-1 were treated and analyzed as described in panel A. The ubiquitinated form of mMcl-1 (wild-type or mutant) is as indicated. The lower panel is a direct immunoblotting analysis of cell lysates (1/20 of that used in the upper panel) with anti-HA antibody.