Journal: The Journal of Biological Chemistry

Article Title: The Streptococcal Collagen-binding Protein CNE Specifically Interferes with α V β 3 -mediated Cellular Interactions with Triple Helical Collagen *

doi: 10.1074/jbc.M110.146001

Figure Lengend Snippet: CNE inhibits PDGF BB-induced α V β 3 -mediated, but not α 2 β 1 -mediated collagen gel contraction. C2C12 cells were seeded in collagen type I gels in the presence or absence of CNE or an inhibitor to α V -integrins. Top panel , C2C12 cells did not contract collagen gels in the absence of exogenous factors. Addition of PDGF-BB at a final concentration of 20 ng/ml induced efficient contraction of C2C12 cells that was abolished by adding a final concentration of 10 μ m function-blocking inhibitor to α V -integrin subunit (αV-inh). Middle panel , addition of CNE at a final concentration of 350 n m nearly completely blocked PDGF BB-induced α V β 3 integrin-mediated contraction. Note that the control and PDGF-BB values in the two upper panels are identical. Bottom panel , in contrast to α V β 3 integrin-mediated contraction, contraction mediated via integrin α 2 β 1 (C2C12-α 2 cells) was carried out efficiently in the absence of any exogenous factors. Addition of CNE at a final concentration of 350 n m did not inhibit this process. In all three panels values represent averages of at least three independent experiments and error bars are S.E.

Article Snippet: The specific α V -integrin inhibitor cyclo-(Arg-Gly-Asp-Phe-Val) was obtained from Bachem (Bubendorf, Switzerland).

Techniques: Concentration Assay, Blocking Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: The Streptococcal Collagen-binding Protein CNE Specifically Interferes with α V β 3 -mediated Cellular Interactions with Triple Helical Collagen *

doi: 10.1074/jbc.M110.146001

Figure Lengend Snippet: MMP activity is not inhibited by CNE and is not required for PDGF BB-induced α V β 3 integrin-mediated contraction. A , 10 μg of collagen type I was preincubated with 2.2 μg of CNE (equimolar ratio) or with buffer only for 1 h at 37 °C. Immediately afterward the mixture was incubated with 25 ng of aminophenyl mercury acetate-activated MMP-1 for the indicated times and reactions were stopped by adding sample buffer. No change in digestion pattern was observed when collagen was pre-treated with CNE. Collagen chains and reaction products are indicated to the right. B , collagen gel contraction was performed as described under “Experimental Procedures.” GM6001 at 10 μ m did not inhibit PDGF BB-induced C2C12 cell-mediated contraction, but substantially potentiated the effect of PDGF-BB. GM6001 had no effect on C2C12-α 2 -mediated contraction. Graphs represent averages of three independent experiments and error bars are S.E.

Article Snippet: The specific α V -integrin inhibitor cyclo-(Arg-Gly-Asp-Phe-Val) was obtained from Bachem (Bubendorf, Switzerland).

Techniques: Activity Assay, Incubation

Journal: Nutrition Journal

Article Title: P38MAP kinase, but not phosphoinositol-3 kinase, signal downstream of glutamine-mediated fibronectin-integrin signaling after intestinal injury

doi: 10.1186/1475-2891-12-88

Figure Lengend Snippet: PI3-K/Akt siganling regulates FN-expression and does not signal downstream of GLN-mediated FN-Integrin osmosignaling after HS. A ) IEC-6 cells were treated for 1 h with either media, GRGDSP (50 μM), or GRGESP (50 μM), prior to GLN treatment (0 mM or 10 mM) under basal (37°C) and stressed conditions (43°C). Procaspase-3, cleaved caspase-3 levels and ß-actin were measured via Western blot and cleaved Caspase-3 levels are presented as fold change ± SEM (n = 3). B ) IEC-6 cells were treated as described in Figure A. Representative Western blots of 3 independent experiments of [S(P) 473 ]Akt and total Akt are shown as mean fold change relative to total Akt ± SEM and normalized to 0 mM GLN. C ) FN levels with/without GLN (10 mM) and LY294002 (25 μM) treatment under unstressed (37°C) or stressed conditions (43°C) were determined by Western blot after 3 h recovery. Densitometric analysis of FN expression as mean fold change relative to 0 mM GLN cells ± SEM (n = 3-8).

Article Snippet: Cells were, then, treated with/without 10 mM GLN for 15 min, with or without prior 1 h treatment with FN-Integrin inhibitor GRGDSP (50 μM, diluted in dH 2 0), inactive control peptide GRGESP (50 μM, diluted in dH 2 0) (Bachem, Torrance, CA), p38MAPK inhibitor SB203580 (10 μM and 30 μM, diluted in DMSO), or PI3-K inhibitor LY294002 (25 μM, diluted in DMSO) and subjected to HS.

Techniques: Expressing, Western Blot