Journal: The Journal of Biological Chemistry

Article Title: A cell-based high-throughput screening method based on a ubiquitin-reference technique for identifying modulators of E3 ligases

doi: 10.1074/jbc.RA118.003822

Figure Lengend Snippet: Cell-based high-throughput URT-Dual-Luciferase Assay system for SMURF1. A, a schematic of the pRUF-RHOB construct. A fusion protein comprised of triple FLAG-tagged RL, UbR48 moiety, triple FLAG-tagged FL, and RHOB is cleaved in vivo by Ubps at the UbR48-RHOB junction to yield equimolars of triple FLAG-tagged RL-UbR48 and triple FLAG-tagged FL-RHOB. The triple FLAG-tagged FL-RHOB is a substrate of SMURF1 and will be degraded in the presence of SMURF1. B, analysis of the steady-state levels of FL-RHOB by immunoblotting. HEK293T cells were transfected with pRUF-RHOB and HA-tagged SMURF1 (HA/SMURF1), WT, or catalytically inactive mutant C699A, as indicated. After overnight treatment with or without 5 μm MG-132, total cell lysates were subjected to immunoblotting using indicated antibodies to determine the steady-state protein levels. C, luciferase assay of FL-RHOB. HEK293T cells were transfected with pRUF-RHOB and HA/SMURF1 WT or C699A, and treated with or without 5 μm MG-132 as in (B) and then applied to Dual-Glo Luciferase Assay to measure the activities of FL and RL. Results were plotted as the ratio of FL activity to RL activity (FL/RL). D and E, evaluation of the screening system. HEK293T cells co-transfected with pRUF-RHOB and WT HA/SMURF1 were treated overnight with DMSO (open circles) and 5 μm MG-132 (filled squares) as negative or positive controls, respectively. Luciferase activities were measured and plotted using either FL activity alone (D) or the FL/RL ratio (E). F and G, the URT system effectively corrects variation of cell numbers. HEK293T cells co-transfected with pRUF-RHOB and WT HA/SMURF1 were seeded with varying number of cells as indicated and treated with DMSO or MG-132 as in (D). Luciferase activities were measured and plotted as FL alone (F) or FL/RL (G).

Article Snippet: The antibodies used were anti-FLAG M2 mAb (Sigma-Aldrich), anti-HA mAb (Santa Cruz Biotechnology, SC7392), anti-ubiquitin P4D1 mAb (Santa Cruz Biotechnology, SC8017), anti-His antibody (Santa Cruz Biotechnology, SC8036), anti-SMURF1 antibody (Abcam, ab57573), anti-RHOB antibody (Santa Cruz Biotechnology, SC180), anti–ZO-1 antibody (EMD Millipore, MABT11), anti-vimentin antibody (BD Biosciences, 550513), anti-SMAD2 mAb (Cell Signaling Technology, 3122S), anti– p -SMAD2 mAb (Cell Signaling Technology, 3108S), anti–β-actin mAb (Santa Cruz Biotechnology, SC47778), and anti-GAPDH mAb (Santa Cruz Biotechnology, SC32233).

Techniques: High Throughput Screening Assay, Luciferase, Construct, In Vivo, Western Blot, Transfection, Mutagenesis, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: A cell-based high-throughput screening method based on a ubiquitin-reference technique for identifying modulators of E3 ligases

doi: 10.1074/jbc.RA118.003822

Figure Lengend Snippet: High-throughput screening for SMURF1 inhibitors. HEK293T cells co-transfected with pRUF-RHOB and WT HA/SMURF1 were treated overnight with 10 μm of each compound from the compound library. DMSO (green circles) and 5 μm MG-132 (red triangles) were used as negative or positive controls, respectively. Activities of FL and RL were measured and results plotted as FL/RL. Solid lines represent the mean and the mean ± 4 × S.D. of all assay points excluding MG-132–treated wells.

Article Snippet: The antibodies used were anti-FLAG M2 mAb (Sigma-Aldrich), anti-HA mAb (Santa Cruz Biotechnology, SC7392), anti-ubiquitin P4D1 mAb (Santa Cruz Biotechnology, SC8017), anti-His antibody (Santa Cruz Biotechnology, SC8036), anti-SMURF1 antibody (Abcam, ab57573), anti-RHOB antibody (Santa Cruz Biotechnology, SC180), anti–ZO-1 antibody (EMD Millipore, MABT11), anti-vimentin antibody (BD Biosciences, 550513), anti-SMAD2 mAb (Cell Signaling Technology, 3122S), anti– p -SMAD2 mAb (Cell Signaling Technology, 3108S), anti–β-actin mAb (Santa Cruz Biotechnology, SC47778), and anti-GAPDH mAb (Santa Cruz Biotechnology, SC32233).

Techniques: High Throughput Screening Assay, Transfection, Drug discovery

Journal: The Journal of Biological Chemistry

Article Title: A cell-based high-throughput screening method based on a ubiquitin-reference technique for identifying modulators of E3 ligases

doi: 10.1074/jbc.RA118.003822

Figure Lengend Snippet: Counterscreen using the N-end rule pathway. A, a schematic of the pRUF–R-ek construct. The pRUF–R-ek is designed similarly to pRUF-RHOB (Fig. 1A) except that triple FLAG-tagged FL-RHOB is replaced by R-ek–FLAG-FL. B, immunoblotting assay of the steady-state levels of R-ek–FL. HEK293T cells were transfected with pRUF–R-ek with or without HA/SMURF1 as indicated. After 3 h treatment with or without 10 μm MG-132, steady-state protein levels were determined by immunoblotting total cell lysates using indicated antibody. C, luciferase assay of R-ek–FL. HEK293T cells were transfected with or without HA/SMURF1 and pRUF–R-ek, and treated with or without MG-132 as in (B). The activities of FL and RL were then measured and plotted as FL/RL. D, counterscreen of selected compounds from the primary screen. HEK293T cells transfected with pRUF–R-ek were treated 6 h with 10 μm of each of the 37 compounds identified in the primary screen. DMSO and MG-132 were used as negative and positive controls, respectively. The effect of each compound on the FL-RHOB/RL or R-ek–FL/RL ratio was plotted relative to the mean of the eight DMSO-treated control wells, respectively. Solid lines represent the mean ± 4 × S.D. of the DMSO controls, as indicated. Arrow and asterisk show the compound that had minimal effect on R-ek–FL/RL ratio.

Article Snippet: The antibodies used were anti-FLAG M2 mAb (Sigma-Aldrich), anti-HA mAb (Santa Cruz Biotechnology, SC7392), anti-ubiquitin P4D1 mAb (Santa Cruz Biotechnology, SC8017), anti-His antibody (Santa Cruz Biotechnology, SC8036), anti-SMURF1 antibody (Abcam, ab57573), anti-RHOB antibody (Santa Cruz Biotechnology, SC180), anti–ZO-1 antibody (EMD Millipore, MABT11), anti-vimentin antibody (BD Biosciences, 550513), anti-SMAD2 mAb (Cell Signaling Technology, 3122S), anti– p -SMAD2 mAb (Cell Signaling Technology, 3108S), anti–β-actin mAb (Santa Cruz Biotechnology, SC47778), and anti-GAPDH mAb (Santa Cruz Biotechnology, SC32233).

Techniques: Construct, Western Blot, Transfection, Luciferase, Control

Journal: The Journal of Biological Chemistry

Article Title: A cell-based high-throughput screening method based on a ubiquitin-reference technique for identifying modulators of E3 ligases

doi: 10.1074/jbc.RA118.003822

Figure Lengend Snippet: HS-152 inhibits SMURF1-mediated ubiquitination and degradation. A, HS-152 inhibits SMURF1-mediated RHOB degradation in a dose-dependent manner. HEK293T cells were transfected with FLAG-tagged RHOB (F/RHOB) and WT or C699A FLAG-tagged SMURF1 (F/SMURF1) as indicated. After overnight treatment with or without different doses of HS-152, total cell lysates were subjected to immunoblotting using indicated antibodies. The steady-state protein levels were quantified using Image Lab software (Bio-Rad) with β-actin as a loading control. Results were plotted in right panel as the levels of F/RHOB in cells co-transfected with WT F/SMURF1 and F/RHOB at each dose of HS-152 treatment relative to the level of F/RHOB in cells transfected with F/RHOB alone and without HS-152 treatment. B, HS-152 inhibits SMURF1-mediated RHOA degradation in a dose-dependent manner. HEK293T cells transfected with indicated FLAG-tagged RHOA (F/RHOA) and F/SMURF1 (WT or C699A) were treated with different doses of HS-152 and subjected to immunoblotting and then quantified and plotted as in (A). C, HS-152 inhibits SMURF1-mediated SMAD1 degradation in a dose-dependent manner. HEK293T cells transfected with indicated FLAG-tagged SMAD1 (F/SMAD1) and F/SMURF1 (WT or C699A) were treated with a different dose of HS-152 and subjected to immunoblotting and then quantified and plotted as in (A). D, HS-152 up-regulates endogenous RHOB levels through SMURF1. HEK293T cells transfected with control shRNA (sh-Con) or shRNA against SMURF1 (sh-SMURF1) and treated 4 h with or without 2 μm HS-152 and then subjected to immunoblotting assay. The lower panel presents quantitative analysis of Western blotting results (mean ± S.D. of three independent experiments). E, HS-152 inhibits SMURF1-mediated RHOB ubiquitination in vitro. FLAG-tagged RHOB (F/RHOB) and His-tagged SMURF1 (His/SMURF1) expressed and purified from bacteria were subjected to an in vitro ubiquitination assay in the absence or presence of different doses of HS-152 as indicated. The reaction products were then subjected to anti-FLAG immunoprecipitation (IP) followed by immunoblotting assay to detect ubiquitin-conjugated RHOB ((Ub)n-RHOB) using an anti-ubiquitin antibody.

Article Snippet: The antibodies used were anti-FLAG M2 mAb (Sigma-Aldrich), anti-HA mAb (Santa Cruz Biotechnology, SC7392), anti-ubiquitin P4D1 mAb (Santa Cruz Biotechnology, SC8017), anti-His antibody (Santa Cruz Biotechnology, SC8036), anti-SMURF1 antibody (Abcam, ab57573), anti-RHOB antibody (Santa Cruz Biotechnology, SC180), anti–ZO-1 antibody (EMD Millipore, MABT11), anti-vimentin antibody (BD Biosciences, 550513), anti-SMAD2 mAb (Cell Signaling Technology, 3122S), anti– p -SMAD2 mAb (Cell Signaling Technology, 3108S), anti–β-actin mAb (Santa Cruz Biotechnology, SC47778), and anti-GAPDH mAb (Santa Cruz Biotechnology, SC32233).

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Software, Control, shRNA, In Vitro, Purification, Bacteria, Immunoprecipitation

Journal: PLoS ONE

Article Title: MicroRNA-21 Exhibits Antiangiogenic Function by Targeting RhoB Expression in Endothelial Cells

doi: 10.1371/journal.pone.0016979

Figure Lengend Snippet: A . Alignment of potential miR-21 binding sites in the 3′UTR of the RhoB mRNA of different species. B . HUVECs were transfected with a precursor of miR-21 (Pre-miR-21) or with a precursor control (Pre-miR-Ctrl) and with a LNA-21 or with a LNA control (LNA Ctrl). The RhoB mRNA level was analyzed after 48 h by qRT-PCR. C–D . Total protein was extracted from HUVECs 48 h post transfection and RhoA, RhoB and RhoC protein levels were measured by Western blotting. ERK1/2 level was analyzed as an internal control. D . Quantification of ( C ). Quantification was performed using ImageJ software. E–F . Measurement of GTPase activity of RhoB. E . Transfected HUVECs were processed for pull-down assays and Western blot analysis with specific antibody to RhoB. F . Quantification of ( E ). Quantification was performed using ImageJ software. G . Transfected HUVECs were seeded onto gelatin-coated slides and analyzed for fluorescence labeling by phalloidin-FITC (green). Nuclei were visualized by DAPI staining (blue). White arrows show areas with less actin stress fibers. Asterisks represent some elongated HUVECs. Arrowhead indicates stress fibers in the center of the cell. Pictures are representative of three independent experiments. Data are means with the SD. *p<0.05 versus corresponding control; (n = 3).

Article Snippet: Blots were blocked for 1 h at room temperature with 8% milk in Tris-buffered saline with 0.1% Tween 20 and probed overnight at 4°C with anti-RhoB, RhoA or RhoC primary antibody (RhoA sc-418; RhoB sc-180, RhoC sc-26480 Santa Cruz Biotechnology) (or for 1 h at room temperature for anti-ERK1,2 M-5670, VCL V-9131, Sigma; Sox7 sc-134024, Santa Cruz Biotechnology; MPRIP ab56164, Abcam and 2 h at 37°C for SPRY1 sc-380048 Santa Cruz Biotechnology).

Techniques: Binding Assay, Transfection, Control, Quantitative RT-PCR, Western Blot, Software, Activity Assay, Fluorescence, Labeling, Staining