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

Figure Lengend Snippet:

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

Journal: International journal of oncology

Article Title: Modulation of the transforming growth factor-beta1-induced Smad phosphorylation by the extracellular matrix receptor beta1-integrin.

doi: 10.3892/ijo_00000463

Figure Lengend Snippet: Figure 2. The expression and the phosphorylation status of Smad2 and Smad3 in the parental, the mock- and ß1A-integrin-transfected HepG2 cells. The cells without TGF-ß1 stimulation were analyzed by Western blotting with anti-Smad2 and anti-Smad3 antibodies. pSmad2C, Smad2 phosphorylated at C-terminal site; pSmad2L, Smad2 phosphorylated at the linker region; tSmad2, total Smad2; pSmad3C, Smad3 phosphorylated at the C-terminal site; pSmad3L, Smad3 phosphorylated at the linker region; tSmad3, total Smad3.

Article Snippet: The antibodies against the phosphorylated form of the C-terminal region of Smad2 (Ser465/467) and Smad3 (Ser423/425), as well as the antibody against the phosphorylated form of the linker region of Smad2 (Ser245/250/255), were purchased from Cell Signaling (Boston, MA).

Techniques: Expressing, Phospho-proteomics, Transfection, Western Blot

Journal: International journal of oncology

Article Title: Modulation of the transforming growth factor-beta1-induced Smad phosphorylation by the extracellular matrix receptor beta1-integrin.

doi: 10.3892/ijo_00000463

Figure Lengend Snippet: Figure 3. The modulation of the TGF-ß1-induced phosphorylation of the Smad proteins in the parental, the mock- and ß1A-integrin-transfected HepG2 cells. The cells were treated with 2 ng/ml of TGF-ß1, were harvested at the indicated times and were analyzed by Western blotting using each antibody. pSmad2C, Smad2 phosphorylated at C-terminal site; pSmad2L, Smad2 phosphorylated at the linker region; tSmad2, total Smad2; pSmad3C, Smad3 phosphorylated at the C-terminal site; pSmad3L, Smad3 phosphorylated at the linker region; tSmad3, total Smad3.

Article Snippet: The antibodies against the phosphorylated form of the C-terminal region of Smad2 (Ser465/467) and Smad3 (Ser423/425), as well as the antibody against the phosphorylated form of the linker region of Smad2 (Ser245/250/255), were purchased from Cell Signaling (Boston, MA).

Techniques: Phospho-proteomics, Transfection, Western Blot

Journal: International journal of oncology

Article Title: Modulation of the transforming growth factor-beta1-induced Smad phosphorylation by the extracellular matrix receptor beta1-integrin.

doi: 10.3892/ijo_00000463

Figure Lengend Snippet: Figure 4. The altered nuclear translocation of the phosphorylated Smad proteins after TGF-ß1 treatment in ß1A-integrin-transfected HepG2 cells. The cells were separated into the cytoplasmic (Cy) and nuclear (Nu) fractions with or without 2 ng/ml of TGF-ß1 for 1 h and were analyzed by Western blotting. Histone H1 was detected as the control for the nuclear proteins. pSmad2C, Smad2 phosphorylated at C-terminal site; pSmad2L, Smad2 phosphorylated at linker region; tSmad2, total Smad2; pSmad3C, Smad3 phosphorylated at the C-terminal site; pSmad3L, Smad3 phosphorylated at the linker region; tSmad3, total Smad3.

Article Snippet: The antibodies against the phosphorylated form of the C-terminal region of Smad2 (Ser465/467) and Smad3 (Ser423/425), as well as the antibody against the phosphorylated form of the linker region of Smad2 (Ser245/250/255), were purchased from Cell Signaling (Boston, MA).

Techniques: Translocation Assay, Transfection, Western Blot, Control

Journal: International journal of oncology

Article Title: Modulation of the transforming growth factor-beta1-induced Smad phosphorylation by the extracellular matrix receptor beta1-integrin.

doi: 10.3892/ijo_00000463

Figure Lengend Snippet: Figure 5. The involvement of the MAP kinase pathway in the alteration of the Smad phosphorylation in the ß1A-integrin-transfected cells. The HepG2 cells overexpressing ß1A-integrin were treated with PD98509 (ERK inhibitor; left), SB203580 (p38 inhibitor; center) and SP600125 (JNK inhibitor; right) for 6 h prior to the 1-h treatment with 2 ng/ml of TGF-ß1. pSmad2C, Smad2 phosphorylated at the C-terminal site; pSmad2L, Smad2 phosphorylated at the linker region; tSmad2, total Smad2; pSmad3C, Smad3 phosphorylated at the C-terminal site; pSmad3L, Smad3 phosphorylated at the linker region; tSmad3, total Smad3.

Article Snippet: The antibodies against the phosphorylated form of the C-terminal region of Smad2 (Ser465/467) and Smad3 (Ser423/425), as well as the antibody against the phosphorylated form of the linker region of Smad2 (Ser245/250/255), were purchased from Cell Signaling (Boston, MA).

Techniques: Phospho-proteomics, Transfection

Journal: International journal of oncology

Article Title: Modulation of the transforming growth factor-beta1-induced Smad phosphorylation by the extracellular matrix receptor beta1-integrin.

doi: 10.3892/ijo_00000463

Figure Lengend Snippet: Figure 6. The restoration of the TGF-ß1-induced Smad-mediated transcription by mutant Smads lacking phosphorylation sites in the linker region. The HepG2 cells were transfected with the SBE4 reporter plasmid and the ß1- integrin expression plasmid together with mutant Smads lacking phosphory- lation sites in the linker region (Smad2-EPSM and Smad3-EPSM) were then treated with 2 ng/ml of TGF-ß1 for 24 h. The data are shown as the mean ± SD from three independent experiments. *P<0.05, **P<0.01 compared with control without Smad2-EPSM and Smad3-EPSM.

Article Snippet: The antibodies against the phosphorylated form of the C-terminal region of Smad2 (Ser465/467) and Smad3 (Ser423/425), as well as the antibody against the phosphorylated form of the linker region of Smad2 (Ser245/250/255), were purchased from Cell Signaling (Boston, MA).

Techniques: Mutagenesis, Phospho-proteomics, Transfection, Plasmid Preparation, Expressing, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis

doi: 10.1161/atvbaha.111.240622

Figure Lengend Snippet: Figure 1. Vigorous angiogenic sprouting is accompanied by defective pericyte attachment in the absence of heparan sulfate (HS). Wild type (wt), exostosin 1 (Ext1)/, and N-deacetylase/N-sulfotransferases (Ndst)1/2/ embryoid bodies (EBs) were grown in collagen I for 12 days (A–G) or 17 days (H) in the presence of vascular endothelial growth factor A (VEGFA; 30 ng/mL). Endothelial cells (ECs) were visualized by staining for CD31 (green) (A, D), and binary images were used for quantification (B, E). ECs and pericytes (PCs) in wt, Ext1/, and Ndst1/2/ EBs were visualized by staining for CD31 (green) and NG2 (red), respectively (C, F, G, and H). Note the absence of pericytes in Ndst1/2/ EBs, and the presence of detached (star), partially detached (arrow heads) and attached (arrows) pericytes in the Ext1/ EBs. The sprout ing area fractions of individual EBs were quantified (I; n12) as well as the number of NG21 cells and their localization in relation to ECs (J, K; n 11). The ability of TGF1 (5 ng/mL) to induce Smad2 phosphorylation (P-Smad2) (L, M; n3) and platelet-derived growth factor B (PDGFB) (100 ng/mL) to induce platelet-derived growth factor -receptor beta (PDGFR) phosphorylation (N) was studied in wt and Ext1/ EBs. PDGFR protein was immunoprecipitated (IP) followed by immunodetection of phosphorylated tyrosine residues (P-Tyr) and PDGFR. (O) Quantification of PDGFR phoshorylation relative to total PDGFR protein after PDGFB stimulation and IP (n2).

Article Snippet: The membranes were incubated with the following primary antibodies: rabbit anti-phospho Smad2 (antibody kindly provided by Dr Aris Moustakas, the Ludwig Institute for Cancer Research, Uppsala, Sweden), mouse anti-Smad1/2/3 (sc- 7960; Santa Cruz), mouse anti-phosphotyrosine (4G10; Upstate, Lake Placid, NY), rabbit polyclonal anti-PDGFRß antibody (sc-432; Santa Cruz), goat anti-human VEGFR2 (AF357; R&D Systems;), goat anti-mouse VEGFR2 (AF644; R&D Systems), rabbit anti-phospho Akt, rabbit anti-phospho-ERK1/2, rabbit anti-total Akt or rabbit anti-total ERK1/2 (all from Cell Signaling Technology).

Techniques: Histone Deacetylase Assay, Staining, Phospho-proteomics, Derivative Assay, Immunoprecipitation, Immunodetection

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis

doi: 10.1161/atvbaha.111.240622

Figure Lengend Snippet: Figure 4. Chondroitin sulfate (CS) binds angiogenic factors and supports TGF- and platelet-derived growth factor B (PDGFB)-signaling. A, Vascular endothelial growth factor A (VEGFA)165, PDGFB, and TGF1 bind to 35S-labeled chondroitin sulfate (CS) isolated from wild type (wt) and Exostosin 1 (Ext1)/ EBs. The graphs illustrate the mean of two measuring points. B, Phospho-AKT (P-AKT) and phospho- ERK1/2 (P-ERK1/2) activation induced by PDGFB in human aortic smooth muscle cells (hAoSMCs) after treatment with heparitinase III (HEPase) or chondroitinase (CSase). C, D, Quantification of the results shown in (B). E, Smad2 phosphorylation induced by TGF1 in hAoSMCs after treatment with HEPase or CSase. F, Quantification of P-Smad2 after TGF1 stimulation. G, Activation of platelet-derived growth factor-receptor (PDGFR) by PDGFB in N-deacetylase/N-sulfotransferases (Ndst1)/ mouse embryonic fibroblasts. Treatment with CSase leads to reduced activation of the receptor. n3 for all signaling experiments.

Article Snippet: The membranes were incubated with the following primary antibodies: rabbit anti-phospho Smad2 (antibody kindly provided by Dr Aris Moustakas, the Ludwig Institute for Cancer Research, Uppsala, Sweden), mouse anti-Smad1/2/3 (sc- 7960; Santa Cruz), mouse anti-phosphotyrosine (4G10; Upstate, Lake Placid, NY), rabbit polyclonal anti-PDGFRß antibody (sc-432; Santa Cruz), goat anti-human VEGFR2 (AF357; R&D Systems;), goat anti-mouse VEGFR2 (AF644; R&D Systems), rabbit anti-phospho Akt, rabbit anti-phospho-ERK1/2, rabbit anti-total Akt or rabbit anti-total ERK1/2 (all from Cell Signaling Technology).

Techniques: Derivative Assay, Labeling, Isolation, Activation Assay, Phospho-proteomics, Histone Deacetylase Assay