Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 6

1.
Fig. 3.

Fig. 3. From: E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1).

Specificity of E2 requirement for in vitro ubiquitinylation with recombinant GST-ARD1. Standard ubiquitinylation assays contained 0.8 pmol of E1, 2.2 pmol of GST-ARD1 (Upper) or GST (Lower), and 0.4 μg of the indicated human E2 ubiquitin-conjugating enzyme. Ubiquitinylated proteins were detected with anti-ubiquitin antibodies; arrowhead indicates ubiquitinylated UbcH6 (E2). The experiment was repeated twice with similar results. Activities of UbcH1 and UbcH3/CDC34 were demonstrated by the production of E2-ubiquitin conjugates that were stable under reducing conditions (data not shown).

Alessandro Vichi, et al. Proc Natl Acad Sci U S A. 2005 February 8;102(6):1945-1950.
2.
Fig. 1.

Fig. 1. From: E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1).

ARD1 and its mutant forms: structure and SDS/PAGE of recombinant GST-proteins. (Upper) Predicted domain structures of ARD1, a TRIM/RBCC family member, and related GST-tagged recombinant proteins (6, 9). (Lower) Samples (0.5 μg) of purified recombinant GST proteins were subjected to SDS/PAGE in 4–20% Tris-glycine gels and detected by silver staining. The doublet at 26 kDa probably represents GST generated via proteolysis of bacterially synthesized GST-fusion proteins and was present in all GST-ARD1 preparations.

Alessandro Vichi, et al. Proc Natl Acad Sci U S A. 2005 February 8;102(6):1945-1950.
3.
Fig. 2.

Fig. 2. From: E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1).

GST-ARD1-catalyzed formation of ubiquitinylated proteins in vitro required E1 and E2. Samples (2.2 pmol) of native or heat-inactivated (asterisk) GST-ARD1 (1–574), GST-cytohesin-1 (C-1), or GST were incubated with 0.8 pmol of E1 and/or 16 pmol of E2 (UbcH6) in standard ubiquitinylation assays (total volume of 30 μl) for 60 min at 30°C before separation of proteins by SDS/PAGE. Blots were reacted with anti-ubiquitin monoclonal antibody. Dots indicate positions of mono- or diubiquitin; the arrowhead indicates ubiquitinylated E2. Data were replicated at least three times.

Alessandro Vichi, et al. Proc Natl Acad Sci U S A. 2005 February 8;102(6):1945-1950.
4.
Fig. 5.

Fig. 5. From: E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1).

Requirement of intact ARD1 RING finger domain for E3 ubiquitin ligase activity. (Top Left) Ubiquitinylation activity of GST-ARD1 or the indicated mutant proteins (5 pmol each). (Right) Standard assays (30 μl) contained, as indicated, 0.8 pmol of E1 and 16 pmol of UbcH6 (E2), with 2.2 pmol of the indicated recombinant GST-ARD1 or GST protein. (Middle Left) Standard assays contained, as indicated, His-tagged ARD1 protein (9 pmol) or GST-ARD1 (2.2 pmol) with E1 and/or E2; E1/E2 lane contained no ARD1. In Bottom Left, the same blot reacted with anti-ARD1 antibodies is showing unmodified GST- and His-ARD1. Arrowheads indicate ubiquitinylated E2. Data were replicated at least twice.

Alessandro Vichi, et al. Proc Natl Acad Sci U S A. 2005 February 8;102(6):1945-1950.
5.
Fig. 4.

Fig. 4. From: E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1).

Ubiquitinylation catalyzed by E1, E2, and GST-ARD1 (E3) as a function of time and enzyme concentrations. (Left) Standard assay mixture (270 μl) containing 75 nM ARD1 (2.2 pmol/30 μl) was incubated at 30°C with 26 nM E1 (0.8 pmol/30 μl) and 0.53 μM UbcH6 (E2, 16 pmol/30 μl). At the indicated times, 30-μl samples were removed and added to 10 μl of 4× Laemmli sample buffer. (Center) Standard assays (30 μl) containing 0, 0.1, 0.2, 0.4, 0.8, or 1.6 pmol of GST-ARD1 or 2.2 pmol of GST were incubated for 60 min. (Right) Assays (30 μl) containing 2.2 pmol of GST-ARD1 with the indicated amounts (pmol) of E1 and E2 were incubated for 60 min. Data in Left and Center were replicated at least twice.

Alessandro Vichi, et al. Proc Natl Acad Sci U S A. 2005 February 8;102(6):1945-1950.
6.
Fig. 6.

Fig. 6. From: E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1).

Ubiquitinylation of ARD1, free GST, and UbcH6 (E2) in vitro. Standard assay mixture (150 μl) containing 75 nM GST-ARD1 (2.2 pmol/30 μl) (A) or 300 nM His-tagged ARD1 (9 pmol/30 μl) (B and C) was incubated at 30°C with 26 nM E1 (0.8 pmol/30 μl), 0.53 μM UbcH6 (E2, 16 pmol/30 μl), and 3.9 μM recombinant human ubiquitin (117 pmol/30 μl) or mutant human ubiquitin in which arginine replaced all lysines (asterisks) (see methods regarding mutant ubiquitin concentration). At the indicated times, 30-μl samples were removed and added to 10 μl of 4× Laemmli sample buffer. (A) Blot was reacted with anti-GST antibodies before stripping and reaction with anti-ubiquitin antibodies. Free GST was present in all GST-ARD1 preparations, because it was bound along with GST-ARD1 to glutathione-Sepharose during purification. (B and C) Reactions contained His-tagged ARD1 and either wild-type or lysine-free ubiquitin (B, asterisk), or only lysine-free ubiquitin (C). Blots were reacted with anti-ubiquitin, anti-ARD1, or anti-UbcH6 antibodies as indicated. Arrow and arrowheads indicate, respectively, the unmodified and modified forms of the UbcH6 (E2). Data were replicated twice.

Alessandro Vichi, et al. Proc Natl Acad Sci U S A. 2005 February 8;102(6):1945-1950.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk