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1.
Figure 6

Figure 6. From: SCFFBXL15 regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1.

Smurf2 is also targeted for ubiquitination and degradation by FBXL15. (A) Interaction analysis of Nedd4 family E3s with FBXL15 by Co-IP assays in HEK293T cells. (B) Nedd4 family members were individually cotransfected together with increasing amounts of FBXL15 into HEK293T cells. Their expression levels were determined. (C) Ubiquitination levels of Nedd4 family members by FBXL15 were determined through in vivo ubiquitination assays. (D) FBXL15 WT, but not the indicated mutants, promotes Smurf2 degradation in a proteasome-dependent manner. (E) HEK293T cells were transfected with Flag-FBXL15, Flag-FBXL15-ΔF, or Flag-FBXL15-F-box together with Smurf2 and HA-ALK4 before 3TP luciferase activity was measured. Data are mean±s.d. (n=3). (F) Knockdown of endogenous FBXL15 inhibits ALK4-induced 3TP-luc activity. Data are mean±s.d. (n=3). Asterisk means P<0.05.

Yu Cui, et al. EMBO J. 2011 July 6;30(13):2675-2689.
2.
Figure 4

Figure 4. From: SCFFBXL15 regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1.

FBXL15 promotes Smurf1 degradation by targeting the sites of Smurf1 K355 and K357. (A) A series of Smurf1 deletion mutants were generated (represented in the upper panel) and coexpressed with increasing amounts of FBXL15. Protein levels of Smurf1 wild type and deletions were detected (lower). (B) Smurf1 3KR mutation in the WH linker appears to resist degradation by FBXL15. Smurf1 wild type and the indicated mutant were coexpressed with increasing amounts of FBXL15. The Smurf1 protein levels were determined. (C) The indicated Smurf1 individual mutants in the WH linker were coexpressed with or without FBXL15. The Smurf1 protein levels were determined and quantified by densitometric analysis. (D) HEK293T cells transfected with the indicated plasmids were treated with CHX for the indicated time and then harvested and analysed by western blot. Quantification was performed and each point represents the mean±s.d. for triplicate experiments. (E) Smurf1 2KR mutation attenuates FBXL15-mediated ubiquitination of Smurf1. The ubiquitination assays were performed.

Yu Cui, et al. EMBO J. 2011 July 6;30(13):2675-2689.
3.
Figure 7

Figure 7. From: SCFFBXL15 regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1.

fbxl15 is expressed in zebrafish embryos and required for dorsoventral development. (AE) The expression pattern of fbxl15 in zebrafish embryos at indicated stages, detected by whole-mount in situ hybridization. Lateral views: (A′–E′) indicate sense probe. (FI) Knockdown of fbxl15 caused dorsalized phenotypes. Wild-type embryos were injected with morpholinos at one- to two-cell stages and observed at 24 hpf. Quantification result is also shown. (JN) Knockdown of fbxl15 altered dorsal (gsc) and ventral (eve1 and gata2) markers expression at the shield stage. Injection doses: fbxl15-MO1 (J′–L′) or fbxl15-cMO1 (J–L), 7.5 ng; fbxl15 mRNA (J″–L″), 50 pg. Dorsal views with animal pole to the top for gsc; animal views with dorsal to the right for eve1; lateral views with dorsal to the right for gata2. Statistical data and qRT–PCR results for maker changes in (JL″) are shown in (M) and (N). Asterisk means P<0.05. (OV) Overexpression or knockdown of fbxl15 affects the stability of Smurf-GFP proteins. The smurf1Mt is a mutant form with K355R and K357R for smurf1. Wild-type embryos were injected at one- to two-cell stages with indicated synthetic mRNAs or MOs and observed at mid-gastrulation stages. Injection doses: 200 pg for smurf1-gfp, smurf1Mt-gfp or smurf2-gfp mRNA; 300 pg for fbxl15 mRNA; 7.5 ng for fbxl15-MO1; and 12.5 ng for fbxl15-MO2.

Yu Cui, et al. EMBO J. 2011 July 6;30(13):2675-2689.
4.
Figure 3

Figure 3. From: SCFFBXL15 regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1.

FBXL15 promotes Smurf1 ubiquitination. (A) FBXL15 promotes Smurf1 ubiquitination in vivo. HEK293T cells were cotransfected with Flag-Smurf1 (WT or CA) and HA-Ub, with or without Myc-FBXL15. Ubiquitinated Smurf1 were immunoprecipitated with an anti-Flag antibody and protein A/G-agarose beads under denaturing conditions to eliminate any Smurf1-associated proteins through non-covalent bonds. (B) Knockdown of endogenous Cullin1, Roc1, or FBXL15 decreased Smurf1 ubiquitination. HEK293T cells were transfected with indicated siRNAs and HA-Ub. Smurf1 ubiquitination were immunoprecipitated and detected with Smurf1 antibody. (C) To determine the efficiency of Smurf1 ubiquitination by FBXL15 in the context of different E2s, the semi-in vitro ubiquitination system was performed with two purified E2s, UbcH5c, and UbcH7, in addition to HA-Ub, E1, purified cell-expressed Flag-FBXL15, and purified bacteria-expressed His-Smurf1. The reaction was incubated at 30°C for 1.5 h and terminated with sample buffer before analysis with anti-Smurf1 antibody. (D) SCFGST-FBXL15 complex increases the ubiquitination of Smurf1 in vitro. The in vitro ubiquitination reactions were performed with the purified HA-Ub, E1, E2 (UbcH5c), purified bacteria-expressed His-Smurf1 (WT or CA), and purified GST-FBXL15 or GST/Cul1/Roc1/Skp1 elution (the Cul1/Roc1/Skp1 were expressed as Myc-tagged proteins in HEK293T cells and the cell lysate was pull down by GSH beads), or GST-FBXL15/Cul1/Roc1/Skp1 complex, as indicated. (E) FBXL15 has no effect on Smurf1 auto-ubiquitination. The in vitro ubiquitination were performed with the purified HA-Ub, E1/E2, purified bacteria-expressed His-Smurf1 (WT or CA) and purified GST-FBXL15. GST-CKIP-1 was used as a positive control.

Yu Cui, et al. EMBO J. 2011 July 6;30(13):2675-2689.
5.
Figure 8

Figure 8. From: SCFFBXL15 regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1.

FBXL15 regulates bone homoeostasis in adult rats. (A) The rat osteoblast-like UMR106 cells were transfected with non-targeting siRNA (NC), vehicle control (VC), and three independent siRNAs against rat FBXL15. The efficiency of FBXL15 knockdown was determined by qRT–PCR to analyse the mRNA levels of FBXL15. (BF) MicroCT analysis of trabecular bone at proximal tibia in five groups of rats. Thirty 6-month-old female Sprague–Dawley rats were divided into the siRNA (#1) treatment group (FBXL15 RNAi group), the non-targeting siRNA control group (NC group), the vehicle control group (VC group), the age-matched group (AM group) and the baseline group (BL group) as described in the Materials and methods. The following parameters bone mineral density (BMD), relative bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were analysed, and data are presented as mean±s.d. Asterisk means P<0.05. (G) Representative three-dimensional reconstruction images of trabecular bone at the proximal tibia from the baseline group (Bl group), AM group, non-targeting siRNA control group (NC group), vehicle control group (VC group), and FBXL15 siRNA treatment group (RNAi group). (H) Schematic representation of Smurf1 regulation by SCFFBXL15 complex.

Yu Cui, et al. EMBO J. 2011 July 6;30(13):2675-2689.
6.
Figure 5

Figure 5. From: SCFFBXL15 regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1.

FBXL15 attenuates the effect of Smurf1 on BMP signalling. (A) Myc-Smads 1, 3, 5, and constant amounts of Flag-Smurf1 with increasing amounts of Flag-FBXL15 were transfected into HEK293T cells, followed by immunoblotting analysis. (B) HepG2 cells were stimulated with BMP-2 (50 ng/ml) for 1 h in the absence or presence of FBXL15 siRNA. Cells were harvested at the indicated times after BMP removal, and total cell lysates were analysed. (C) HEK293T cells were transfected with the FBXL15 WT or deletion mutants together with Smurf1, as indicated. Thirty-six hours after transfection, cells were treated with BMP-2 (100 ng/ml) for 12 h before BRE luciferase activity was measured. Data are mean±s.d. (n=3). (D) Effects of FBXL15 on BMP signalling activity by Smurf1 mutants. Reporter activities of BRE-luc in HEK293T cells transfected with a series of Smurf1 mutants, Flag-FBXL15, as indicated, were measured after treatment with BMP-2. Data are mean±s.d. (n=3). (E) Knockdown of endogenous Cullin1, Roc1, or FBXL15 inhibits BMP signalling activity. BMP signalling activity in HepG2 cells was measured by BRE luciferase reporter gene assay. Data are mean±s.d. (n = 3). (F) Cells were transfected with siRNA against FBXL15 or non-targeting control siRNA and, 48 h later, treated with BMP-2 (100 ng/ml) or left untreated for 2 h. Expression of BMP target genes ID1, Smad6 was analysed by qRT–PCR. Asterisk means P<0.05.

Yu Cui, et al. EMBO J. 2011 July 6;30(13):2675-2689.
7.
Figure 1

Figure 1. From: SCFFBXL15 regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1.

Smurf1 interacts with FBXL15 both in vitro and in cultured cells. (A) The ‘bait' and the ‘prey' regions of Smurf1 and FBXL15 in yeast two-hybrid screening are shown schematically. (B) Direct interaction between FBXL15 and Smurf1 was revealed by GST pull-down assays. Both input and pull-down samples were subjected to immunoblotting with anti-His and anti-GST antibodies. Input represents 20% of that used for pull down. (C) Co-immunoprecipitation (Co-IP) of endogenous FBXL15 and endogenous Smurf1 from HEK293 cells. Western-blot analysis of whole-cell lysates and immunoprecipitates with Smurf1 antibody or control IgG. To avoid Smurf1 degradation, the proteasome inhibitor MG132 was added and incubated for 12 h before the cells were harvested. IP, immunoprecipitate; IB, immunoblotting; IgG, immunoglobulin; IgG HC, IgG heavy chain. (D) FBXL15 was co-immunoprecipitated with both wild type and catalytic mutant forms of Smurf1. HEK293T cells transfected with Myc-FBXL15 and Flag-Smurf1 (WT or C699A) were immunoprecipitated with anti-Flag, followed by immunoblotting analysis. (E) FBXL15 was colocalized with Smurf1 within the cytoplasm. Myc-FBXL15 and Flag-Smurf1 were cotransfected into MCF7 cells and 24 h later stained with mouse anti-Flag and rabbit anti-Myc antibodies before visualization by confocal microscopy. To avoid Smurf1 degradation, MG132 was added and incubated for 12 h before the cells were harvested. (F) FBXL15 interacts with Smurf1 through its LRR domains. Flag-Smurf1 and FBXL15 deletion mutants were transfected into HEK293T cells, and Co-IP assays were performed. Asterisks indicate FBXL15 deletion mutants. (G, H) Mapping of interacting regions of Smurf1 with FBXL15. Flag-FBXL15 and the indicated Smurf1 truncates were coexpressed in HEK293T cells. Cell lysates were incubated with anti-Flag to precipitate Smurf1 deletion mutants. Both the lysates and the immunoprecipitates were analysed by western blot with the indicated antibodies. Asterisks indicate Smurf1 deletion mutants. HECT-N-lobe-L/S, the large/small subdomain of Smurf1 HECT N-lobe. (I) Schematic representation of the binding regions between Smurf1 and FBXL15.

Yu Cui, et al. EMBO J. 2011 July 6;30(13):2675-2689.
8.
Figure 2

Figure 2. From: SCFFBXL15 regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1.

FBXL15 forms a functionally active SCF complex and promotes the proteasomal degradation of Smurf1. (A) FBXL15 is associated with Skp1, Cullin1, and Roc1. HEK293T cells transfected with empty vector, Flag-FBXL15 or Flag-FBXL15-ΔF were immunoprecipitated with anti-Flag, and then whole-cell lysates and immunoprecipitates were subjected to immunoblotting with anti-Cullin1, anti-Skp1, and anti-Roc1 antibodies. (B) FBXL15 expression decreases the steady-state level of Smurf1 WT and C699A mutant. HEK293T cells were transfected with a constant amount of Smurf1 (WT or CA) and increasing amounts of FBXL15. After 24 h, cells were treated with MG132. Aliquots of total lysates were immunoblotted to detect Smurf1 and FBXL15. (C) Wild-type FBXL15, but not FBXL15 deletions, promote Smurf1 degradation. HEK293T cells were transfected with Flag-Smurf1 and various FBXL15 mutants. Cells were treated with or without MG132 for 12 h and harvested to detect Smurf1. (D) Knockdown of Cullin1, Roc1, or FBXL15 stabilizes endogenous Smurf1. HEK293T cells were transfected with two independent siRNA duplexes against Cullin1 (1# and 2#), Roc1 (1# and 2#), FBXL15 (2# and 3#), or non-targeting control siRNA. Cell lysates were analysed by immunoblotting with the indicated antibodies. Relative intensity of the Smurf1 bands is indicated. (E) Knockdown of FBXL15 increases the half-life of endogenous Smurf1. HEK293T cells were transfected with control siRNA, 2# or 3# siRNAs against FBXL15 or cotransfected with FBXL15 siRNA-resistant mutants. Thirty-six hours after transfection, cells were treated with cycloheximide (50 μg/ml) for the indicated times and then harvested for immunoblotting. (F) Smurf1 degradation is specific to FBXL15. HEK293T cells were transfected with the indicated Myc-tagged F-box protein constructs together with Flag-Smurf1. Cell lysates were harvested and analysed by immunoblotting. (G) HeLa cells transfected with control siRNA or FBXL15 siRNA were synchronized by thymidine-aphidicolin (2 mM) treatment. At the indicated time after release, the indicated proteins were analysed by immunoblotting.

Yu Cui, et al. EMBO J. 2011 July 6;30(13):2675-2689.

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