Journal: The Journal of biological chemistry

Article Title: Definition of an unexpected ligand recognition motif for alphav beta6 integrin.

doi: 10.1074/jbc.274.4.1979

Figure Lengend Snippet: FIG. 1. Integrin preparation and adsorption. Panel A, SDS-poly- acrylamide gel electrophoresis (7.5% gel) under nonreducing conditions of transmembrane truncated recombinant avb3 (lane 1), placental avb5 (lane 2), transmembrane truncated recombinant avb6 (lane 3), and platelet aIIbb3 (lane 4). The gel is stained with Coomassie Blue. Ar- rowheads and numbers to the right indicate the migration position of marker proteins and their molecular mass in kDa. The band at ;65 kDa in lane 4 may be a serum albumin contaminant. Panel B, adsorp- tion control. Integrins avb3-DTM (solid circles), avb6-DTM (open cir- cles), and avb5 (solid triangles down) were adsorbed to 96-well plates from 1.5-mg/ml solutions and blocked as for phage display screens and ligand competition assays. Biotinylated antibody 17E6 (27), serially diluted as indicated, was added, and after incubation and washing bound antibody was detected with anti-biotin-alkaline phosphatase conjugate. The non-av integrin control, aIIbb3, gave background bind- ing of 17E6 (A405 , 0.05) (data not shown).

Article Snippet: The integrin avb3-DTM, avb5, and avb6-DTM preparations were ;95% pure as judged by anti-integrin ELISA2 using a and b chainspecific monoclonal antibodies (data not shown) and by SDS-polyacrylamide gel electrophoresis against molecular weight standards (Bio-Rad).

Techniques: Adsorption, Polyacrylamide Gel Electrophoresis, Recombinant, Staining, Migration, Marker, Control, Incubation

Journal: Journal of the American Society of Nephrology

Article Title: Enhancer of Zeste Homolog 2 Inhibition Attenuates Renal Fibrosis by Maintaining Smad7 and Phosphatase and Tensin Homolog Expression

doi: 10.1681/asn.2015040457

Figure Lengend Snippet: Figure 1. 3-DZNeP inhibits renal fibroblast activation in a dose-dependent manner. (A and E) Normally cultured NRK-49F cells were treated with 3-DZNeP (0–10 mM) for 36 hours. Then, cell lysates were prepared and subjected to immunoblot analysis with antibodies against (A) a-SMA, collagen I, fibronectin, or GAPDH or (E) EZH2, H3K27me3, or GAPDH. The levels of (B) fibronectin, (C) type 1 collagen, (D) a-SMA, or (F) EZH2 and H3K27me3 were quantified by densitometry and normalized with GAPDH. Values are the means6SDs of at least three independent experiments. Bars with different letters (a–d) for each molecule are significantly different from one an- other (P,0.05).

Article Snippet: Antibodies to fibronectin, collagen I (A2), EGFR, TGFb-RI, Smad7, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). aVb6, phospho-Smad3, and Smad3 antibodies were pur- chased from Abcam, Inc. (Cambridge, MA).

Techniques: Activation Assay, Cell Culture, Western Blot

Journal: Journal of the American Society of Nephrology

Article Title: Enhancer of Zeste Homolog 2 Inhibition Attenuates Renal Fibrosis by Maintaining Smad7 and Phosphatase and Tensin Homolog Expression

doi: 10.1681/asn.2015040457

Figure Lengend Snippet: Figure 2. 3-DZNeP inhibits renal fibroblast activation in a time-dependent manner. (A and E) Normally cultured NRK-49F cells were treated with 10 mM 3-DZNeP for the indicated time. Then, cell lysates were prepared and subjected to immunoblot anal- ysis with antibodies against (A) a-SMA, collagen I, fibronectin, or GAPDH or (E) EZH2, H3K27me3, or GAPDH. The levels of (B) fibronectin, (C) type 1 collagen, (D) a-SMA, or (F) EZH2 and H3K27me3 were quantified by densitometry and normalized with GAPDH. Values are the means6SDs of at least three independent experiments. Bars with different letters (a–d) for each molecule are significantly different from one an- other (P,0.05).

Article Snippet: Antibodies to fibronectin, collagen I (A2), EGFR, TGFb-RI, Smad7, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). aVb6, phospho-Smad3, and Smad3 antibodies were pur- chased from Abcam, Inc. (Cambridge, MA).

Techniques: Activation Assay, Cell Culture, Western Blot

Journal: Journal of the American Society of Nephrology

Article Title: Enhancer of Zeste Homolog 2 Inhibition Attenuates Renal Fibrosis by Maintaining Smad7 and Phosphatase and Tensin Homolog Expression

doi: 10.1681/asn.2015040457

Figure Lengend Snippet: Figure 3. Treatment with 3-DZNeP or GSK126 inhibits TGFb1–induced renal fibroblast activation. Serum–starved NRK-49F cells were pretreated with (A–C) 3-DZNeP (10 mM) or (D–G) GSK126 (0–5 mM) for 1 hour and then exposed to TGFb1 (2 ng/ml) for an additional 24 hours. (A and D) Cell lysates were prepared and subjected to immunoblot analysis with antibodies against fibronectin, collagen I, a-SMA, EZH2, H3K27me3, or GAPDH. Expression levels of (B and E) fibronectin, collagen I, and a-SMA or (C, F, and G) EZH2 and H3K27me3 were quantified by densitometry and normalized with GAPDH. Values are the means6SDs of at least three independent experiments. Bars with different letters (a–f) are significantly different from one another (P,0.05).

Article Snippet: Antibodies to fibronectin, collagen I (A2), EGFR, TGFb-RI, Smad7, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). aVb6, phospho-Smad3, and Smad3 antibodies were pur- chased from Abcam, Inc. (Cambridge, MA).

Techniques: Activation Assay, Western Blot, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Enhancer of Zeste Homolog 2 Inhibition Attenuates Renal Fibrosis by Maintaining Smad7 and Phosphatase and Tensin Homolog Expression

doi: 10.1681/asn.2015040457

Figure Lengend Snippet: Figure 4. Knockdown of EZH2 with siRNA inhibits renal fibroblast activation. Serum–starved NRK-49F cells were transfected with siRNA targeting EZH2 or scrambled siRNA and then, incubated in (A–C) 5% FBS or (D–F) TGFb1 (2 ng/ml) for an additional 24 hours. (A and D) Cell lysates were prepared for immunoblot analysis with antibodies against fibronectin, collagen I, a-SMA, EZH2, H3K27me3, tubulin or GAPDH. (B, C, E, and F) Expression levels of a-SMA, collagen I, fibronectin, EZH2, or H3K27me3 were quantified by densitometry and normalized with tubulin or GAPDH. Values are the means6SDs of at least three independent experiments. Bars with different letters (a–d) for each molecule are significantly different from one another (P,0.05). FN, fibronectin.

Article Snippet: Antibodies to fibronectin, collagen I (A2), EGFR, TGFb-RI, Smad7, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). aVb6, phospho-Smad3, and Smad3 antibodies were pur- chased from Abcam, Inc. (Cambridge, MA).

Techniques: Knockdown, Activation Assay, Transfection, Incubation, Western Blot, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Enhancer of Zeste Homolog 2 Inhibition Attenuates Renal Fibrosis by Maintaining Smad7 and Phosphatase and Tensin Homolog Expression

doi: 10.1681/asn.2015040457

Figure Lengend Snippet: Figure 5. Administration of 3-DZNeP attenuates development of renal fibrosis and deposition of ECM in obstructed kidneys. (A) Photomicrographs illustrating Masson tri- chrome staining of kidney tissue (magnification 3200). (B) The Masson trichrome–positive tubulointerstitial area (blue in A) relative to the whole area from ten random cortical fields was analyzed. Data are represented as the means6SDs (n=6). (C) Kidney tissue lysates were subjected to immunoblot analysis with antibodies against a-SMA, collagen 1, fi- bronectin, or tubulin. Expression levels of fibronectin, collagen 1, a-SMA, or tubulin were quantified by densitometry, and the levels of (D) fibronectin, (E) collagen 1, and (F) a-SMA were normalized with tubulin. Values are the means6SDs (n=6). Means with different letters (a–c) are significantly different from one another (P,0.05).

Article Snippet: Antibodies to fibronectin, collagen I (A2), EGFR, TGFb-RI, Smad7, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). aVb6, phospho-Smad3, and Smad3 antibodies were pur- chased from Abcam, Inc. (Cambridge, MA).

Techniques: Staining, Western Blot, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Enhancer of Zeste Homolog 2 Inhibition Attenuates Renal Fibrosis by Maintaining Smad7 and Phosphatase and Tensin Homolog Expression

doi: 10.1681/asn.2015040457

Figure Lengend Snippet: Figure 10. Treatment with SF1670, a specific PTEN inhibitor, abolishes the inhibitory effect of 3-DZNep on renal interstitial fi- broblast activation in NRK-49F cells. Normally cultured NRK-49F cells were pretreated with 2 mM SF1670 for 1 hour, and then exposed to 10 mM 3-DZNeP for 24 hours. Cell lysates were prepared and subjected to immunoblot analysis with antibodies against (A) a-SMA and fibronectin; (C) phospho-EGFR, EGFR, phospho-PDGFRb, and PDGFRb; (E) PTEN, phospho-AKT, and AKT; or (A and E) GAPDH. (B, D, and F) The levels of a-SMA, fibronectin, phospho-EGFR, EGFR, phospho-PDGFRb, PDGFRb, PTEN, phospho-AKT, and AKT were quantified by densitometry and normalized with GAPDH, EGFR, PDGFR, or AKT as indicated. Values are the means6SDs of at least three independent ex- periments. Bars with different letters (a–d) for each molecule are significantly different from one another (P,0.05).

Article Snippet: Antibodies to fibronectin, collagen I (A2), EGFR, TGFb-RI, Smad7, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). aVb6, phospho-Smad3, and Smad3 antibodies were pur- chased from Abcam, Inc. (Cambridge, MA).

Techniques: Activation Assay, Cell Culture, Western Blot