Journal: iScience

Article Title: Long noncoding RNA Lnc-DIF inhibits bone formation by sequestering miR-489-3p

doi: 10.1016/j.isci.2022.103949

Figure Lengend Snippet:

Article Snippet: Ser465/Ser467 phosphorylated SMAD2 Rabbit polyclonal antibody , Bioss , Cat# bs-3419R, RRID: AB_10880886.

Techniques: Luciferase, Plasmid Preparation, Recombinant, In Vivo, Transfection, Reporter Assay, In Situ Hybridization, Sequencing, Software

Journal: Brain : a journal of neurology

Article Title: Integrin control of the transforming growth factor-β pathway in glioblastoma.

doi: 10.1093/brain/aws351

Figure Lengend Snippet: Figure 3 Integrin inhibition decreases Smad2 phosphorylation and TGF-b-induced reporter gene activity. (A) Whole cell protein lysates of LN-308 glioma cells, cultured with specific antibodies to different integrins as indicated (top), or transiently transfected with small interfering RNA oligonucleotides specific for one integrin or a control small interfering RNA (middle), were assessed for pSmad2 and total Smad2/3 levels by immunoblot, using GAPDH as a loading control. In the bottom panel, LN-308, LN-18, U87MG, T98G or LNT-229 cells were exposed to control peptide () or cilengitide (10 mM) as indicated and assessed for pSmad2 and total Smad2/3 levels. (B) LN-308 or LNT-229 cells were exposed to blocking antibodies (top) or small interfering RNA oligonucleotides (middle) as in (A). In the bottom panel, LN-308 or LNT-229 cells were exposed to the control peptide RAD (black bars), 2.5 mM (light grey) or 10 mM (dark grey) cilengitide for 24 h. Intracellular TGF-b signalling was assessed by reporter assays using the pGL3-SBE4-Luc and pGl2-3TP-Luc constructs (*P 5 0.05 and **P 5 0.01 compared with isotype control and scrambled small interfering RNA or RAD, respectively).

Article Snippet: TGF-b1, phospho-Smad2 (Ser465/467) (clone 138D4) and Smad2 (clone 86F7) antibodies were purchased from Cell Signaling Technology.

Techniques: Inhibition, Phospho-proteomics, Activity Assay, Cell Culture, Transfection, Small Interfering RNA, Control, Western Blot, Blocking Assay, Construct

Journal: Brain : a journal of neurology

Article Title: Integrin control of the transforming growth factor-β pathway in glioblastoma.

doi: 10.1093/brain/aws351

Figure Lengend Snippet: Figure 6 Effects of cilengitide on Smad2 phosphorylation in LN-308 gliomas in vivo. (A) LN-308 glioma cells were exposed to RAD or cilengitide (10 mM), TGF-b2 (10 ng/ml) or SD-208 (1 mM) and assessed for pSmad2 expression after 48 h by immunofluorescence microscopy (original magnification 40). (B) LN-308 cells were inoculated intracerebrally in athymic CD1 nude mice. The animals were treated intraperitoneally with cilengitide (90 mg/g body weight) or PBS for 5 days starting 3 weeks after tumour cell inoculation. Subsequently, the animals’ brains were removed, shock-frozen and integrin expression was assessed by immunohistochemistry as indicated (original magnification 10 for haematoxylin and eosin and 40 for pSmad2). (C) pSmad2 levels were assessed by immu- nohistochemistry (C) and quantified (D).

Article Snippet: TGF-b1, phospho-Smad2 (Ser465/467) (clone 138D4) and Smad2 (clone 86F7) antibodies were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, In Vivo, Expressing, Microscopy, Immunohistochemistry

Journal: PLoS ONE

Article Title: Long-Term Gene Therapy with Thrombospondin 2 Inhibits TGF-β Activation, Inflammation and Angiogenesis in Chronic Allograft Nephropathy

doi: 10.1371/journal.pone.0083846

Figure Lengend Snippet: In gene therapy, treated rats with renal transplants TGF-β were evaluated by immunohistochemistry. Active TGF-β was either detected directly by using an antibody recognizing active TGF-β (A) or indirectly by evaluation of phosphorylation of the TGF-β signaling molecule smad2/3 (B) or expression of the TGF-β downstream target PAI-1 within the glomeruli (C) or the cortex (D). Representative microphotographs of PAI-1 staining in renal grafts treated with control (E) and TSP-2 overexpressing plasmid (F) are shown. Total TGF-β1 (G) and TGF-β2 (H) was similar in both groups. Control (n = 8) vs. TSP-2 treated (n = 8); *p<0,05.

Article Snippet: The TGF-β system was studied using antibodies to TGF-β1 (rabbit anti-human TGF-β1, Santa Cruz Biotechnology Inc. ); TGF-β2 (rabbit anti-human TGF-β2, Santa Cruz ); active TGF-β1 (chicken anti-human active TGF-β1, (R&D systems, Germany , , Phospho-Smad2/3 (rabbit anti-human Smad2 peptide phosphorylated at Ser-433/435, Santa Cruz ) or PAI-1, a rabbit polyclonal to human plasminogen activator inhibitor-1 (Santa Cruz).

Techniques: Immunohistochemistry, Phospho-proteomics, Expressing, Staining, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: Long-Term Gene Therapy with Thrombospondin 2 Inhibits TGF-β Activation, Inflammation and Angiogenesis in Chronic Allograft Nephropathy

doi: 10.1371/journal.pone.0083846

Figure Lengend Snippet: Representative microphotographs from immunohistological staining of kidney grafts for active TGF-β (A, control plasmid; B, TSP-2 therapy, brown cytosolic staining), P-smad 2/3 (C, control plasmid; D, TSP-2 therapy, brown nuclear staining), PAI-1 (E, control plasmid; F, TSP-2 therapy, brown staining), fibronectin (G, control plasmid; H, TSP-2 therapy, brown staining) and alpha-smooth muscle actin (I, control plasmid; J, TSP-2 therapy, brown staining) are shown.

Article Snippet: The TGF-β system was studied using antibodies to TGF-β1 (rabbit anti-human TGF-β1, Santa Cruz Biotechnology Inc. ); TGF-β2 (rabbit anti-human TGF-β2, Santa Cruz ); active TGF-β1 (chicken anti-human active TGF-β1, (R&D systems, Germany , , Phospho-Smad2/3 (rabbit anti-human Smad2 peptide phosphorylated at Ser-433/435, Santa Cruz ) or PAI-1, a rabbit polyclonal to human plasminogen activator inhibitor-1 (Santa Cruz).

Techniques: Staining, Control, Plasmid Preparation

Journal: Human reproduction (Oxford, England)

Article Title: Activin A regulates trophoblast cell adhesive properties: implications for implantation failure in women with endometriosis-associated infertility.

doi: 10.1093/humrep/deq097

Figure Lengend Snippet: Figure 2 pSmad2 of HTR8 trophoblast cells.(A) Treatment of HTR8 trophoblast cells with activin A (ActA; 1, 10, 50 and 100 ng/ml) for 24 h results in a dose-dependent increase in pSmad2 as shown by western blot. Addition of activin inhibitor, SB431542 (SB: 10 mM) in conjunction with ActA (50 ng/ml), decreased pSmad2 compared with ActA alone. The lower band represents total Smad2 which is not changed with ActA treatment. (B) Densitometric analysis of representative western blot in (A). Each bar represents the mean of two experiments assayed in duplicate.

Article Snippet: Membranes were exposed to autoradiography film (Hyperfilm ECL; GE Healthcare) for 5 min. Membranes were then washed in stripping buffer (Re Blot Plus, by guest on N ovem ber 16, 2015 http://hum rep.oxfordjournals.org/ D ow nloaded from Chemicon International, CA, USA) before blocking again in 10% skim milk/TBS, overnight incubation with rabbit anti-human Smad2 (86F7; Cell Signalling Technology, #3122; 1:1000 in 5% skim milk/TBS) and secondary antibody and detection as described above.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Hexokinase 2, Glycogen Synthase and Phosphorylase Play a Key Role in Muscle Glycogen Supercompensation

doi: 10.1371/journal.pone.0042453

Figure Lengend Snippet: Fold change in glycogenin normalized by total protein content of 1 hour (▪) or 24 hours (▪) stimulated samples compared to control situation (□) measured by Western-blot of Glycogenin-1. The inset shows a representative blot for each situation (from 0 to 24 hours of rest): in the upper panel samples with 1 hour of CLFS, and the lower panel the groups with 24 hours of CLFS. C is the contralateral non-stimulated control sample of each sample showed. In the bar chart the control group was composed with the pool of all contralateral1 non-stimulated muscle samples results (n = 50). n = 5 for the stimulated and rested groups. Data are means ± SE. There was no statistical difference under any condition.

Article Snippet: The primary antibodies used were: muscle GS (mouse monoclonal from Chemicon, dilution 1∶2000), glycogenin (guinea pig polyclonal, dilution 1∶2000), AMPK-P (T172) (rabbit polyclonal from Cell Signalling, dilution 1∶1000), and AMPKα1 or AMPKα2 (goat polyclonal, dilution 1∶2000).

Techniques: Control, Western Blot

Journal: Cell reports

Article Title: Modeling Progressive Fibrosis with Pluripotent Stem Cells Identifies an Anti-fibrotic Small Molecule

doi: 10.1016/j.celrep.2019.11.019

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Article Snippet: Rabbit p-SMAD2/3 , R and D systems , Cat# MAB8935; RRID:AB_2313773.

Techniques: Virus, Recombinant, Luciferase, Imaging, Enzyme-linked Immunosorbent Assay, Hydroxyproline Assay, Software