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