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Results: 4

1.
Figure 2

Figure 2. From: The CRY box: a second APCcdh1-dependent degron in mammalian cdc20.

Characterization of the CRY degron in cdc20. (A,B) Cdc20–Venus complementary RNA constructs (A) were expressed in oocytes; cycloheximide (CHX) was then added to block further protein translation. (B) Cdc20174−216 (n=13) and cdc20184−216 (n=17; black traces) were stable, whereas cdc20154−216 (n=20) and cdc20164−216 (n=15; red traces) were degraded. The cdc20 sequence became stable when residues 164–174 were removed. a and b denote significantly different fluorescence at the 16 h time point (P<0.05 χ2 test). (C) To examine which residues were crucial for degradation in cdc20 residues 164–174, point mutations of each residue were made to alanine, as indicated. Mean loss of fluorescence of green fluorescent protein (GFP) in oocytes after 14 h was expressed as a quotient with respect to cdc20164−216–GFP, such that cdc20164−216 was assigned 100% degradation (n=15–20 per construct). Mutations that are defined as very stable are marked in red.

Alexandra Reis, et al. EMBO Rep. 2006 October;7(10):1040-1045.
2.
Figure 1

Figure 1. From: The CRY box: a second APCcdh1-dependent degron in mammalian cdc20.

Identification of a second degradation signal in cdc20. (A) Germinal-vesicle-arrested oocytes were microinjected with complementary RNA to green fluorescent protein (GFP; black, n=16), cdc20–GFP (red, n=18) or ΔKENcdc20–GFP (blue, n=14). Allowing 2–3 h for expression, oocytes were then incubated with cycloheximide (CHX). a and b denote significantly different fluorescence at the 16 h time point (P<0.05 χ2 test). (B) Various cdc20 constructs, coupled at their carboxyl terminus to GFP, were assayed for their degradation in oocytes on addition of CHX (n=15–25 per construct). Mean GFP fluorescence loss in oocytes after 14 h was expressed as a quotient with respect to cdc20–GFP, such that full-length cdc20 was assigned 100% degradation. From these deletion and mutation experiments, we identified two regions that were determinants of cdc20 stability: the KEN box (in blue) and residues 154–216 in cdc20 (red).

Alexandra Reis, et al. EMBO Rep. 2006 October;7(10):1040-1045.
3.
Figure 4

Figure 4. From: The CRY box: a second APCcdh1-dependent degron in mammalian cdc20.

CRY-box degradation in G1 embryos. (A,B) Four green fluorescent protein (GFP)-containing constructs were used: construct containing both KEN box and CRY box (cdc201−216; n=20; black); construct containing CRY box only (ΔKENcdc201−216; n=22; red); construct containing KEN box only (cdc201−154; n=23; blue); or construct without degron (ΔKENcdc201−154; n=22; grey). Two-cell G1 embryos expressing one of these four constructs were imaged on addition of cycloheximide (CHX). a–c denote significantly different fluorescence at the 16 h time point (P<0.05 χ2 test). (C) Boxed: comparison of the C-box and the CRY-box degron in human cdc20. Residues needed for CRY-box degradation are marked in red. Comparison of the CRY box in human, rat, mouse, Xenopus and Drosophila (asterisk denotes identical residues, colon conserved residues and dot semiconserved residues). The equivalent sequence in budding yeast is also shown. (D) Lack of degradation for the construct C-box cdc20164−216 on addition of CHX at the time indicated (n=19). This construct is similar to cdc20164−216–GFP used previously (Fig 2B), except that 165-CRYIPSL-171 is changed to 165-DRYIPHR-171 to mimic the cdc20 C box.

Alexandra Reis, et al. EMBO Rep. 2006 October;7(10):1040-1045.
4.
Figure 3

Figure 3. From: The CRY box: a second APCcdh1-dependent degron in mammalian cdc20.

Degradation of CRY-box-containing constructs requires APCcdh1 activity. (A) Fluorescence levels of 154−216cdc20–green fluorescent protein (GFP) on cycloheximide (CHX) addition. In some oocytes, methylubiquitin (MeUb; 1 μg/ml) was microinjected or MG132 (50 μg/ml) was added to the culture medium before imaging. a–c denote significantly different fluorescence at the 16 h time point (P<0.05 χ2 test). (B) In vitro-translated cdc20–GFP, cdc20154−216–GFP or ΔKENcdc20–GFP was incubated in a ubiquitination mix with or without ubiquitin (Methods; predicted molecular weights are indicated by arrows); in the presence of ubiquitin, proteins show loss of full-length band intensity and gain of high-molecular-weight polyubiquitination adducts (vertical line). (C) Germinal-veside-stage V oocytes were microinjected with cdh1 morpholino (MOcdh1) and cultured for 24 h. A further microinjection of complementary RNA to cdc20154−216–GFP was made and allowing a few hours for expression, at time t=0 h, either CHX was added (n=14; grey trace) or cdh1 cRNA was microinjected (n=21; red trace). a and b denote significantly different fluorescence at the 16 h time point (P<0.05 χ2 test).

Alexandra Reis, et al. EMBO Rep. 2006 October;7(10):1040-1045.

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