Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TSG-6 inhibits neutrophil migration via direct interaction with the chemokine CXCL8

doi: 10.4049/jimmunol.1300194

Figure Lengend Snippet: Interaction between the Link module domain of T6G-6 and the GAG-binding site of CXCL8 inhibits CXCL8-heparin binding and CXCL8-mediated neutrophil transmigration WT or mutant CXCL8 (250 nM) was immobilized onto MaxiSorp plates and b-heparin (0-100 ng/well) (A) or b-heparin (25 ng/well) in combination with a range of concentrations of rhTSG-6 or Link_TSG6 (0-1000 nM) (B) , or in combination with a range of concentrations of WT Link_TSG6, Link_TSG6_D or Link_TSG6_T (0-2000 nM) (C) was added in the fluid phase. Binding of b-heparin was detected using Extravidin alkaline phosphatase followed by disodium p -nitrophenyl phosphate; absorbance at 405 nm was determined after 5 min (A) or 10 min (B,C) . Data are plotted as mean values ( n = 8) ± SEM and fitted using Origin Pro (v8). K D values of 5 ± 12 nM and 26 ± 11 nM were determined for the binding of b-heparin to WT CXCL8 and CXCL8_D, respectively (A) . IC 50 values of ( B ) 81 ± 18 nM and 71 ± 14 nM for the inhibition of b-heparin binding to CXCL8 by rhTSG-6 and Link_TSG6, respectively, and (C) 105 ± 5 nM, 172 ± 3 nM and 45 ± 5 nM for the inhibition of this interaction by WT Link_TSG6, Link_TSG6_D and Link_TSG6_T, respectively, were determined. In (D) , migration of differentiated HL-60 cells across an EA.hy 926 cell monolayer was determined in response to CXCL8 (3.6 nM (+)) alone or in combination with 5:1 molar ratios of WT Link_TSG6, Link_TSG6_D or Link_TSG6_T ( n = 8). Data are plotted as mean values (± SEM) relative to non-stimulated controls (−), where ** and *** = p

Article Snippet: Bound b-heparin was detected by addition of Extravidin-Alkaline Phosphatase (1:10,000; Sigma) followed by SIGMAFAST™ p -nitrophenyl phosphate solution (200 μl/well; Sigma).

Techniques: Binding Assay, Transmigration Assay, Mutagenesis, Inhibition, Migration

Journal: Bioscience of Microbiota, Food and Health

Article Title: Isolation of lactic acid bacteria bound to the porcine intestinal mucosa and an analysis of their moonlighting adhesins

doi: 10.12938/bmfh.16-012

Figure Lengend Snippet: Detection of adhesins in PBS extracts by a receptor overlay (RO) analysis. After SDS-PAGE using 1 mg/ml PBS extracts, proteins were electrophoretically transferred to a PVDF membrane. The membrane was blocked with 5% skim milk. After washing, the membrane was soaked in TBS-T containing the biotinylated PIM (A), BSA (control protein) (B), or only TBS-T buffer (negative control) (C) overnight. After washing, protein bands were visualized using ExtrAvidin Alkaline Phosphatase Conjugate and ECF substrate with a luminescent image analyzer (excitation, 440 nm; emission, 560 nm). M: biotinylated molecular weight marker. The numbers (shows arrows) indicate proteins that were identified ( Table 2 ).

Article Snippet: After washing with TBS-T, ExtrAvidin Alkaline Phosphatase Conjugate (Sigma-Aldrich) was added, followed by incubation for 30 min. After washing with TBS-T, ECF Substrate for Western Blotting (GE Healthcare, Tokyo, Japan) was added, and visualization was performed using an LAS-3000 luminescent image analyzer (Fujifilm, Tokyo, Japan) (excitation, 440 nm; emission, 560 nm).

Techniques: SDS Page, Negative Control, Molecular Weight, Marker