Journal: Nature communications

Article Title: OTUB1 enhances TGFβ signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3.

doi: 10.1038/ncomms3519

Figure Lengend Snippet: Figure 1 | OTUB1 interacts with TGFb-stimulated SMAD2/3/4. (a) HEK293 cells stably expressing GFP-only, GFP-SMAD1 or GFP-SMAD3 were treated with 50 pM TGFb or 25 ng ml 1 BMP for 1 h before lysis in the presence of dithiobis[succinimidyl propionate] (DSP). GFP immunoprecipitates (IPs) were separated by SDS-PAGE and interacting partners identified by mass spectrometry. (b) An endogenous IP with OTUB1 antibody or pre-immune sheep IgG was performed in HaCaTcell extracts, stimulated without or with 50 pM TGFb or 25 ng ml 1 BMP for 1 h. Cell extracts (input), endogenous IgG or OTUB1 IPs and the corresponding immune-depleted flow-through extracts (O1 ¼ OTUB1-depleted untreated HaCaT extract) were resolved by SDS-PAGE and immunoblotted (IB) with the indicated antibodies. (c) As in b, except that a time course of 50 pM TGFb treatment was performed for up to 6 h before lysis. (d) An endogenous IP with anti-OTUB1 antibody or pre-immune sheep IgG was performed in extracts from bone marrow-derived macrophages (BMDMs) and mouse embryonic fibroblasts (MEFs) stimulated with or without 50 pM TGFb for 1 h. Before TGFb stimulation, BMDMs were serum starved for 2 h, whereas MEFs were not starved. Cell extracts (input) or IPs were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. SMAD-LP, linker-phosphorylated SMAD; SMAD-TP, tail-phosphorylated SMAD.

Article Snippet: Anti-phospho-Ser423/425 SMAD3 (SMAD3-TP; number 600-401-919) and anti-phospho-Thr179 SMAD3 (SMAD3-LP; number 600-401-C48S) were purchased from Rockland.

Techniques: Stable Transfection, Expressing, Lysis, SDS Page, Mass Spectrometry, Derivative Assay

Journal: Nature communications

Article Title: OTUB1 enhances TGFβ signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3.

doi: 10.1038/ncomms3519

Figure Lengend Snippet: Figure 3 | Depletion of OTUB1 represses TGFb-induced transcription. (a) C2C12 cells were transfected with three different siRNAs (#1, #2 and #3) targeting mouse OTUB1 (300 pM/10-cm dish each) and lysed 48 h after transfection. Extracts were resolved by SDS-PAGE and immunoblotted with OTUB1 and tubulin antibodies. (b) C2C12 cells stably expressing a SMAD3-dependent TGFb-responsive CAGA-luciferase reporter construct were transfected with iFoxO4 or iOTUB1#1. Cells were treated with or without 50 pM TGFb for 6 h before lysis and luciferase activity was measured. Data are represented as mean and error bars indicate s.d. (n ¼ 3). (c) HaCaTcells stably expressing shRNA against OTUB1 or transfected with control ( ) or OTUB1 siRNA (300 pM/10-cm dish each) for 48 h were lysed, and extracts were resolved by SDS-PAGE and immunoblotted with OTUB1 and GAPDH antibodies. (d) HaCaT cells, transfected with human OTUB1 siRNA, human FoxO4 siRNA, or stably expressing OTUB1 shRNA, were treated with 50 pM TGFb for 4 h before RNA isolation. Relative expression levels of indicated mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. (n ¼ 6). (e) HaCaT cells depleted of human OTUB1 or FoxO4 by RNAi were treated with 50 pM TGFb for 1 h. TGFb was then washed off and SB505124 (1 mM) added. RNA was isolated 45 min after TGFb removal. Relative expression levels of OTUB1 and CTGF mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. (n ¼ 6). Statistical significance of differences between experimental groups was assessed with Student’s t-test. **Po0.01 and ***Po0.001.

Article Snippet: Anti-phospho-Ser423/425 SMAD3 (SMAD3-TP; number 600-401-919) and anti-phospho-Thr179 SMAD3 (SMAD3-LP; number 600-401-C48S) were purchased from Rockland.

Techniques: Transfection, SDS Page, Stable Transfection, Expressing, Luciferase, Construct, Lysis, Activity Assay, shRNA, Control, Isolation, Quantitative RT-PCR

Journal: Nature communications

Article Title: OTUB1 enhances TGFβ signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3.

doi: 10.1038/ncomms3519

Figure Lengend Snippet: Figure 4 | Characterization of OTUB1 activity in vitro (a) A schematic representation of OTUB1 indicating the positions of key residues and domains. (b,c) Human recombinant GST-OTUB1 or indicated GST-OTUB1 mutants were incubated with K48-linked di-ubiquitin chains in a DUB assay buffer for 1 h at 30 C. The reaction was stopped by the addition of 1 SDS sample buffer and the assay mix was resolved by SDS-PAGE and immunoblotted with GST or ubiquitin antibodies as indicated. (d–f) HEK293 cells were co-transfected with vectors encoding N-terminal HA-tagged OTUB1 or indicated HA-OTUB1 mutants (C91S, DN, D88A/C91S/H265A (D/C/H), H265A, K71R) and N-terminal FLAG-tagged SMAD3. Before lysis (in the presence of iodoacetamide), cells were treated with or without 50 pM TGFb and 10 mM bortezomib for 3 h. FLAG immunoprecipitates (IP) or extracts were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.

Article Snippet: Anti-phospho-Ser423/425 SMAD3 (SMAD3-TP; number 600-401-919) and anti-phospho-Thr179 SMAD3 (SMAD3-LP; number 600-401-C48S) were purchased from Rockland.

Techniques: Activity Assay, In Vitro, Recombinant, Incubation, Ubiquitin Proteomics, SDS Page, Transfection, Lysis

Journal: Nature communications

Article Title: OTUB1 enhances TGFβ signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3.

doi: 10.1038/ncomms3519

Figure Lengend Snippet: Figure 5 | OTUB1 prevents SMAD3 ubiquitylation in vitro. (a) For in-cell polyubiquitylation of FLAG-SMAD2/3/4, vectors encoding FLAG-SMAD2/3/4 were co-transfected with HA-NEDD4L and HA-ubiquitin in HEK293 cells and treated with 50 pM TGFb and 10 mM bortezomib for 3 h before lysis and FLAG-SMAD2/3/4 were immunoprecipitated. An in vitro DUB assay of in-cell polyubiquitylated FLAG-SMAD2/3/4 was performed with GST-OTUB1 and the indicated GST-OTUB1 mutants in DUB assay buffer for 1 h at 30 C. The assay mix was resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (b) An in vitro ubiquitylation assay was performed with human recombinant SMAD2. SMAD2 was incubated with E1, E2, E3 and ubiquitin in ubiquitylation assay buffer for 1 h at 30 C. Increasing concentrations of GST-OTUB1 and GST-OTUB1 C91S (8–60 ng ml 1) were added at the start of the ubiquitylation assay (time 0). Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (c) FLAG-SMAD2/3/4 IPs (from HEK293 cells expressing FLAG-SMAD2/3/4 treated with 50 pM TGFb for 1 h before lysis) were ubiquitylated in vitro in ubiquitylation assay buffer for 1 h at 30 C using E1, E2, E3 and ubiquitin. The indicated DUBs were added at the start of the ubiquitylation assay (time 0) and proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (d) HaCaT cells were stably transfected with vectors encoding siRNA-resistant silent mutations (rescue) of the indicated OTUB1 constructs. These cells were then transfected with control FoxO4 or OTUB1 siRNA for 48 h and treated with or without TGFb for 4 h before RNA isolation. Relative expression levels of indicated mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. (n ¼ 6). Statistical significance of differences between experimental groups was assessed with Student’s t-test. **Po0.01 and ***Po0.001.

Article Snippet: Anti-phospho-Ser423/425 SMAD3 (SMAD3-TP; number 600-401-919) and anti-phospho-Thr179 SMAD3 (SMAD3-LP; number 600-401-C48S) were purchased from Rockland.

Techniques: In Vitro, Transfection, Ubiquitin Proteomics, Lysis, Immunoprecipitation, SDS Page, Ubiquitin Assay, Recombinant, Incubation, Expressing, Stable Transfection, Construct, Control, Isolation, Quantitative RT-PCR

Journal: Nature communications

Article Title: OTUB1 enhances TGFβ signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3.

doi: 10.1038/ncomms3519

Figure Lengend Snippet: Figure 6 | OTUB1 interacts with E2 enzymes and inhibits efficient ubiquitin transfer from E2 to E3. (a) An endogenous IP with OTUB1 antibody or pre-immune sheep IgG was performed in HaCaT cell extracts stimulated with or without 50 pM TGFb for 1 h before lysis in the presence of dithiobis[succinimidyl propionate] (DSP). Cell extracts (input), endogenous IgG or OTUB1 IPs and the corresponding immune-depleted flow-through extracts were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (b) An in vitro ubiquitylation assay was performed with human recombinant SMAD3, His-E1 (0.1 mM), ubiquitin, His-E3 (1 mM) and increasing concentrations of GST-E2 (0.1–5 mM) in ubiquitylation assay buffer for 1 h at 30 C. GST-OTUB1 was added at the start of the ubiquitylation assay (time 0). Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (c) His-E1, E2, FLAG-ubiquitin, GST-OTUB1 and mutants were mixed with or without His-E3 and SMAD3 before the addition of ATP. After 10 min at 30 C, proteins were separated by SDS-PAGE and visualized with Coomassie Instantblue.

Article Snippet: Anti-phospho-Ser423/425 SMAD3 (SMAD3-TP; number 600-401-919) and anti-phospho-Thr179 SMAD3 (SMAD3-LP; number 600-401-C48S) were purchased from Rockland.

Techniques: Ubiquitin Proteomics, Lysis, SDS Page, In Vitro, Ubiquitin Assay, Recombinant

Journal: Cell reports

Article Title: Discovery of a Small-Molecule BMP Sensitizer for Human Embryonic Stem Cell Differentiation.

doi: 10.1016/j.celrep.2016.04.066

Figure Lengend Snippet: Figure 6. PD407824 Increases BMP Sensitivity by Depleting p21 and Activating CDK9, which Then Phosphorylates the SMAD2/3 Linker Region, Leading to Decreased Levels of SMAD2/3 Protein and Enhanced Levels of Nuclear SMAD1 (A) PD407824 treatment increases the binding of SMAD1 and SMAD4 proteins indicated by co-immunoprecipitation. (B) PD407824 causes depletion of SMAD2/3 protein levels, measured by western blotting; SMAD2/3 protein levels are normalized to beta-ACTIN protein levels. (C) PD407824 induces the phosphorylation of the SMAD2/3 linker region (T179 for SMAD3). The phosphorylation level was normalized to total levels of SMAD2 protein. (D–F) Flavopiridol, a CDK inhibitor, blocks the PD407824 induced increased Id2 transcript expression (D), upregulated T179 phosphorylation of SMAD3 and decreased SMAD2/3 total protein (E), and increased binding of SMAD1 and SMAD4 proteins indicated by co-immunoprecipitation (F). (G) Knockout of CDK9 blocks the synergistic effect of PD407824. (H) PD407824 depletes p21 protein levels, shown by western blotting. The p21 protein levels are normalized to b-actin protein levels. (I) Relative Id2 transcript expression in p21+/ and p21/ cells. Uninfected cells (wild-type [wt]) and cells infected with sgRNA targeting GFP were used as negative controls. See also Figure S4.

Article Snippet: The antibodies were rabbit anti-phospho-SMAD1/5/9 (Cell Signaling Technology, 9511), rabbit anti-SMAD1 XP (Cell Signal, 6944), rabbit anti-SMAD2/3 (Cell Signal, 3102), rabbit anti-p21 (C-19 antibody; Santa Cruz Biotechnology, sc379), rabbit anti-phospho-SMAD3 T179 (Rockland, 600-401-C48S), and mouse anti-b-actin (Sigma-Aldrich, A1978).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Phospho-proteomics, Expressing, Knock-Out, Infection

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Astragaloside IV alleviates lung inflammation in Klebsiella pneumonia rats by suppressing TGF-β1/Smad pathway

doi: 10.1590/1414-431x2023e12203

Figure Lengend Snippet: Figure 5. A, Expression of transforming growth factor (TGF)-b1 in lung tissues in K. pneumoniae rat model treated with astragaloside IV (AS-IV) or TGF-b1 inhibitor SB-431542 detected by immunohistochemistry (scale bar 50 mm); B, Expression levels of proteins related with TGF-b1/Smad, p-Smad2/Smad2, and p-Smad3/Smad3 pathways in lung determined by western blot. Data are reported as means± SD (n=3). *Po0.05, **Po0.01 compared with Control group; #Po0.05, ##Po0.01 compared with K. pneumoniae group (ANOVA).

Article Snippet: The primary antibodies were: TGF-b1 polyclonal antibodies (1:800, SAB4502954, Sigma-Aldrich), p-Smad2 polyclonal antibodies (1:100, orb451161, Biorbyt, China), Smad2 polyclonal antibodies (1:100, orb507656, Biorbyt), p-Smad3 polyclonal antibodies (1:100, orb313112, Biorbyt), Smad3 polyclonal antibodies (1:100, orb94696, Biorbyt), NF-kB p-p65 polyclonal antibodies (1:100, orb501839, Biorbyt), NF-kB p65 polyclonal antibodies (1:100, orb344389, Biorbyt), IkBa polyclonal antibodies (1:1000, 4812, Cell Signaling Technology, China), p-IkBa polyclonal antibodies (1:800, 2859, Cell Signaling Technology), and GAPDH polyclonal antibodies (1:1000,5174, Cell Signaling Technology).

Techniques: Expressing, Immunohistochemistry, Western Blot, Control