Figure 3 | Cell Death & Disease

Figure 3

From: Cdc20 directs proteasome-mediated degradation of the tumor suppressor SMAR1 in higher grades of cancer through the anaphase promoting complex

Figure 3

Degradation of SMAR1 by Cdc20 is D-box dependent. (a) Schematic representation of D-box location in SMAR1. First D-box is located in 40–43aa and second D-box in 290–293aa location. Schematic diagram showing mutation created in both the D-boxes. (b) Cdc20-mediated SMAR1 degradation is D box dependent. FLAG-SMAR1-WT or FLAG-SMAR1-D1 or FLAG-SMAR1-D2 were co-transfected either with vector or HA-Cdc20 in MCF7 cells. Whole-cell lysates were immunoblotted with indicated antibodies. (c) MCF7 cells were transfected with FLAG-SMAR1-WT and FLAG-SMAR1-D1 and FLAG-SMAR1-D2 mutant. Transfected cells were pulsed with cycloheximide (40 μg/ml) and chased for indicated time points. Expression level of SMAR1-WT, SMAR1-D1 and SMAR1-D2 and tubulin on immunoblot were quantified densitometrically and presented graphically after normalization at zero time point. (d) Cdc20 fails to interact with SMAR1 D-box mutants. MCF7 cells were co-transfected with FLAG-SMAR1-WT, FLAG-SMAR1-D1, FLAG-SMAR1-D2 and HA-tagged Cdc20. Cell lysate was immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins

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