Journal: PLoS ONE

Article Title: Functional Insights into Recombinant TROSPA Protein from Ixodes ricinus

doi: 10.1371/journal.pone.0076848

Figure Lengend Snippet: I. ricinus TROSPA protein-coated ELISA microplates were incubated with the serial dilutions of rabbit TROSPA antiserum or rabbit preimmune antiserum. After washing, the plates were treated with B. garinii OspA protein (C = 30 µg/ml, 100 µl/well), which was subsequently detected by FITC-conjugated anti -Borrelia goat polyclonal IgG. The X axis represents serial sera dilutions, and the Y axis represents the level of fluorescence counts. The level of TROSPA-OspA binding was determined for ten rabbit TROSPA antiserum or preimmune serum concentrations. The level of OspA binding was determined as the arithmetic mean of fluorescence counts based on 16 reactions performed for every TROSPA antiserum or preimmune serum dilution (for details see Materials and methods). The reduction of fluorescence counts was statistically significant (p<0,01) for all serum dilutions except the highest one.

Article Snippet: TROSPA-coated ELISA microplates (prepared as described above) were incubated for 90 min at room temperature with 100 µl/well serially diluted (in PBST) rabbit TROSPA antiserum or rabbit preimmune serum (Eurogentec).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Fluorescence, Binding Assay

Journal: Science Advances

Article Title: Discovery of anti-inflammatory physiological peptides that promote tissue repair by reinforcing epithelial barrier formation

doi: 10.1126/sciadv.abj6895

Figure Lengend Snippet: ( A ) Schema of anti-JIPs Ab administration to Lgr5-EGFP-IRES-creERT2/tdTomato mice with DSS-induced colitis at the recovery stage. ( B ) Immunofluorescence of ZO-1 and DAPI in colon sections from mice treated as in (A). Scale bar, 20 μm. Similar results were obtained in two independent experiments. ( C ) DAI in accordance with assessment of stool consistency and fecal blood in ICR mice, in which 2% DSS was used for recovery ( n = 5 mice). ( D ) Quantification of the numbers of Gr-1–positive cells (9 to 12 images from five mice) treated as described in (C). ( E ) Relative intestinal permeability measured by plasma leakage of FITC-dextran (4 kDa) treated as described in (C) ( n = 5 mice from ctrl + preimmune or Ab, DSS + vehicle, n = 6 mice from ctrl + vehicle, recover + preimmune or Ab). ( F ) Silver staining of recombinant hA1AT incubated with or without MMP-1, MMP-8, or MMP-9. Similar results were obtained in two independent experiments. ( G ) Quantification of relative claudin-1 intensity at cell-cell boundaries of A431 cells treated with the indicated products or control HBSS ( n = 5 images from independent two samples). Similar results were obtained in two independent experiments. See fig. S7E. ( H ) Quantification of the relative claudin-1 intensity at cell-cell boundaries of A431 cells treated with CCM prepared from control, DSS-treated mice, or DSS-recovery mice in the presence or absence of GM6001, or control HBSS for 3 hours ( n = 5 images from independent two samples). Similar results were obtained in two independent experiments. See fig. S7F. Tukey’s test (C, D, G, and H) and the two-tailed t test (E). * P < 0.05, *** P < 0.001.

Article Snippet: Anti-JIP serum or preimmune serum was transfected into A431 cells by ProteoCarry (Funakoshi) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Permeability, Silver Staining, Recombinant, Incubation, Two Tailed Test

Journal: Science Advances

Article Title: Discovery of anti-inflammatory physiological peptides that promote tissue repair by reinforcing epithelial barrier formation

doi: 10.1126/sciadv.abj6895

Figure Lengend Snippet: ( A ) Immunofluorescence of rabbit IgG and ZO-1 in A431 cells transfected with anti-JIPs Ab or preimmune. Transfected cells were treated with JIP m35 (20 μM) or HBSS for 3 hours. Scale bar, 20 μm. ( B ) Liposome cosedimentation assay using biotinylated JIP m35 peptides or IgG (20 nmol each), which were bound to streptavidin-FITC ( n = 3 independent samples). ( C ) Relative barrier permeability measured by paracellular tracer flux analysis using FITC-dextran (4 kDa) in A431 cells transfected with G 12 and/or G 13 siRNA. See fig. S10B ( n = 3 independent samples). ( D ) TER measurements of EpH4 cells transfected with G 12 and/or G 13 siRNAs. See fig. S10D ( n = 3 independent samples). ( E ) Pull-down assay of A431 cells treated with biotinylated-JIP m35 , biotinylated-JIP m35-mut2 , or buffer using streptavidin-Sepharose. Precipitates were immunoblotted with anti-G 12 (upper), G 13 (bottom) antibodies. Asterisk indicates a nonspecific band. Quantitative value of each band was presented. ( F ) Measurement of G 13 activation by JIP m35 , JIP m35-mut1 , and JIP m35-mut2 ( n = 3 independent samples). JIP m35 was prepared at the indicating concentrations. ( G ) Measurement of G protein (G 13 , G i2 , G s , and G q ) activation by JIP m35 ( n = 3 independent samples). ( H ) Immunofluorescence of occludin, GFP, and G 13 in A431 cells transfected with GFP, G 13 , or G13Q226L. Scale bar, 10 μm. ( I ) Line scans represent the fluorescence intensity of occludin along the white arrow in (H), and a black arrowhead represents the position of cell junction. ( J ) The value represents the maximum value minus the minimum value of occludin intensity ( n = 9 cells). Data represent means ± SD (F and G); Tukey’s test (B and J) and Dunnett’s test (C, D, F, and G). n.s., not significant. * P < 0.05, ** P < 0.01, *** P < 0.001. Similar results were obtained in two independent experiments in (A) and (E).

Article Snippet: Anti-JIP serum or preimmune serum was transfected into A431 cells by ProteoCarry (Funakoshi) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Transfection, Permeability, Pull Down Assay, Activation Assay, Fluorescence