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. Author manuscript; available in PMC: 2011 Sep 3.
Published in final edited form as: Nature. 2011 Mar 3;471(7336):104–109. doi: 10.1038/nature09732

Figure 3. Fbw7 promotes Mcl-1 ubiquitination and destruction in a GSK3 phosphorylation-dependent manner.

Figure 3

a–c, GSK3 phosphorylation-dependent degradation of Mcl-1 by Fbw7. Immunoblot analysis of 293T cells transfected with the indicated Myc-Mcl-1 and HA-Fbw7 plasmids in the presence or absence of HA-GSK3. A plasmid encoding GFP was used as a negative control for transfection efficiency. Where indicated, the proteasome inhibitor MG132 was added.

d, 293T cells were transfected with the indicated Myc-Mcl-1 constructs together with the HA-Fbw7 and HA-GSK3 plasmids. Twenty hours post-transfection, cells were split into 60 mm dishes, and after another 20 hours, treated with 20 µg/ml cycloheximide (CHX). At the indicated time points, whole cell lysates were prepared and immunoblots were probed with the indicated antibodies.

e, Wild-type (WT) or Fbw7−/− DLD1 cells were treated with 20 µg/ml cycloheximide (CHX). At the indicated time points, whole cell lysates were prepared and immunoblots were probed with the indicated antibodies. Mcl-1 band intensity was normalized to tubulin, then normalized to the t=0 controls.

f, Immunoblot analysis (IB) of whole cell lysates (WCL) and His-tag pull-down of HeLa cells transfected with the indicated plasmids. Twenty hours post-transfection, cells were treated with the proteasome inhibitor MG132 overnight before harvesting. His-tag pull-down was performed in the presence of 8 M urea to eliminate any possible contamination from Mcl-1-associated proteins.

g, Immunoblot analysis of wild-type (WT) or Fbw7−/− DLD1 cells treated with 10 µM adriamycin (ADR) for the indicated durations of time. Mcl-1 band intensity was normalized to tubulin, then normalized to the t=0 controls.