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.
Figure 6

Figure 6. Model for the role of SENP1 in Elk-1 regulation. From: SENP1 participates in the dynamic regulation of Elk-1 SUMOylation.

In the absence of ERK pathway signalling, Elk-1 is kept in a repressive state by SUMO-mediated HDAC-2 recruitment. Following ERK activation, Elk-1 is de-SUMOylated, leading to enhanced transactivation capacity, and SENP1 plays a pivotal role in this process. SRE, serum-response element.

James Witty, et al. Biochem J. 2010 June 1;428(Pt 2):247-254.
2.
Figure 3

Figure 3. SENP1 de-SUMOylates Elk-1 in vitro. From: SENP1 participates in the dynamic regulation of Elk-1 SUMOylation.

(A) A Coomassie-Blue-stained gel showing the non-SUMOylated and SUMOylated recombinant GST–Elk-1 fusion proteins. (B) Western blot analysis of FLAG-tagged SENP1 and SENP3 purified by immunoprecipitation from transfected HEK-293T cells. The broken line indicates where irrelevant lanes were removed. (C) SUMOylated recombinant GST–Elk-1(205–428) was incubated in the presence or absence of FLAG-tagged SENP1 or SENP3 for the times indicated and samples were detected by SYPRO-Ruby staining after SDS/PAGE. The positions of the recombinant Elk-1 protein and its SUMOylated forms are indicated. * Indicates the position of a co-purifying contaminant.

James Witty, et al. Biochem J. 2010 June 1;428(Pt 2):247-254.
3.
Figure 2

Figure 2. Elk-1 interacts with SENP1. From: SENP1 participates in the dynamic regulation of Elk-1 SUMOylation.

(A and B) Co-immunopreciptation analysis of Elk-1(1–428) and SENPs. (A) Elk-1 was transfected in the absence and presence of the indicated WT and catalytically inactive (CS) versions of FLAG-tagged SENP1. Following immunoprecipitation of Elk-1 (IP), bound SENPs were detected by immunoblotting (IB) with an anti-FLAG antibody. Immunoprecipitated proteins are shown in the top two panels and input proteins are shown in the bottom two panels. (B) Elk-1 was transfected in the absence and presence of the indicated catalytically inactive (CS) versions of FLAG-tagged SENPs. Following immunoprecipitation of Elk-1 (IP), bound SENPs were detected by immunoblotting (IB) with an anti-FLAG antibody. Immunoprecipitated proteins are shown in the top two panels and input proteins in the bottom panel. * Indicates non-specific cross-reacting bands in the IP samples. (C) GST-pulldown analysis of GST–Elk-1(205–428) binding to SENPs. The indicated catalytically inactive SENPs were expressed in HEK-293T cells, the lysates bound to recombinant GST or GST–Elk-1 and were detected by IB with an anti-FLAG antibody (top panel). A Coomassie-Blue-stained gel of the input bait proteins is shown in the bottom panel.

James Witty, et al. Biochem J. 2010 June 1;428(Pt 2):247-254.
4.
Figure 1

Figure 1. SENP1 preferentially activates Elk-1. From: SENP1 participates in the dynamic regulation of Elk-1 SUMOylation.

(A) Schematic diagram of the GAL-driven reporter system used to assay Elk-1-mediated transactivation. (B) Immunoprecipitation/Western blot analysis of the expression of the indicated FLAG-tagged SENP constructs. HEK-293T cells were transfected with different SENP expression vectors followed by immunoprecipitation with an anti-FLAG antibody and detection by Western blot analysis with the same antibody. The * represents a band corresponding to the antibody heavy chain. (C and D) Luciferase reporter analysis of the activity of GAL–Elk-1(1–428) constructs in the presence of co-transfected SENP expression constructs. (C) Wild-type (black bars) and K230R/K249R(K2R) mutant (grey bars) Elk-1 constructs were transfected in the absence and presence of the indicated SENP constructs. (D) Wild-type GAL–Elk-1 constructs were transfected in the presence of increasing amounts (0 ng, 125 ng, 250 ng, 500 ng and 1 μg) of the indicated SENP constructs. (E) Wild-type GAL–Elk-1 constructs were transfected in the presence of 1 μg of the indicated WT (black bars) or catalytically dead (mut; grey bars) SENP constructs. In all cases data are presented relative to the activity of either WT or K2R versions of GAL–Elk-1(1–428) in the presence of the control empty vector (taken as 1). Data are the average of two independent experiments, each with triplicate samples.

James Witty, et al. Biochem J. 2010 June 1;428(Pt 2):247-254.
5.
Figure 4

Figure 4. Depletion of SENP1 decreases the transactivation capacity of Elk-1. From: SENP1 participates in the dynamic regulation of Elk-1 SUMOylation.

(A) Schematic diagram of the GAL-driven reporter system used to assay Elk-1-mediated transactivation following ERK pathway activation. (B) RT–PCR analysis of the expression of the indicated SENPs in HeLa cells in the presence of control siRNAs or siRNA duplexes directed against the SENPs indicated. Data are representative of two independent experiments and are the average of two samples, presented relative to the transcript levels in the presence of control siRNA duplexes (taken as 1). (CE) Luciferase reporter analysis of the activity of GAL–Elk-1(1–428) constructs in HEK-293T cells in the presence of co-transfected siRNA duplexes against SENPs. All data are the average of two experiments carried out in duplicate. Note that the axis does not begin at zero to emphasize the changes we observe. (C and D) Individual siRNA duplexes (25 pmol) were transfected in the presence of either WT (C) or mutant K230R/K249R(K2R) (D) forms of Elk-1. Cells were either untreated (grey bars) or treated with PMA for 6 h (black bars). (E) Cells were treated with PMA after transfection with vectors encoding WT (grey bars) or mutant (K2R) (black bars) versions of GAL–Elk-1(1–428) and with siRNA targeting SENP1 (12.5 pmol), and the additional siRNA constructs (12.5 pmol) against different indicated SENPs.

James Witty, et al. Biochem J. 2010 June 1;428(Pt 2):247-254.
6.
Figure 5

Figure 5. Depletion of SENP1 decreases the activation of the Elk-1 target gene c-FOS. From: SENP1 participates in the dynamic regulation of Elk-1 SUMOylation.

(A) Schematic diagram showing the different molecular events occurring on the c-FOS promoter after PMA (SUMO loss) or anisomycin (SUMO retention) stimulation. In both cases, c-FOS promoter activation is still observed. SRE, serum-response element. (B) Western blot analysis of Elk-1 SUMOylation levels. HEK-293T cells were transfected with expression vectors for His-FLAG-tagged Elk-1 and HA-tagged SUMO-2, and His-tagged Elk-1 was pulled down (PD) from lysates from HEK-293T cells treated with PMA or anisomycin for 40 min. Total and SUMOylated Elk-1 were detected by immunoblotting (IB) with anti-FLAG and anti-HA antibodies respectively. (CF) RT–PCR analysis of the expression of c-FOS in HeLa cells in the presence of control GAPDH siRNAs or siRNA duplexes directed against the SENPs indicated. Either an siRNA duplex against a single SENP (25 pmol) or two siRNA duplexes against distinct SENPs (12.5 pmol each) were transfected and cells were either serum-starved (grey bars) or treated with PMA (C and E) or anisomycin (D and F) for 40 min (black bars). Data are the average of duplicate samples and are presented relative to the transcript levels in serum-starved cells in the presence of control GAPDH siRNA duplexes (taken as 1).

James Witty, et al. Biochem J. 2010 June 1;428(Pt 2):247-254.

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