fitc lta  (Vector Laboratories)

Journal: bioRxiv

Article Title: Simultaneous Control of Infection and Inflammation by Keratin-Derived Antibacterial Peptides (KAMPs) Targeting TLRs and Co-Receptors

doi: 10.1101/2021.01.18.427180

Figure Lengend Snippet: ( A-C ) Molecular docking of KAMP-10 and KAMP-18C (ribbon representation) to TLRs and their co-receptors (ribbon and surface representations). Top five models of the peptides (1 to 5: yellow, green, red, magenta, cyan) docked to the receptors are shown. (A) KAMP-10 was predicted to bind to the hydrophobic cavity of MD-2 that is in complex with TLR4 (top) or in the soluble form (bottom). (B-C) KAMP-18C was predicted to bind to hydrophobic pocket at the central domain of TLR2 (B) and at the N-terminal domain of CD14 (C). ( D ) Representative FACS plots (left) and quantification (right) showing the extent of LPS binding to TLR4/MD-2-conjugated beads. Latex beads with human TLR4-MD-2 complex conjugated on the surface were incubated with the peptides (KAMP-10 or SC-10 at 48 μg/ml, or KAMP-18C at 96 μg/ml) in the absence or presence of soluble MD-2 (1 μg/ml), washed, then incubated with FITC-LPS (1 μg/ml). Bead surface TLR4 was labeled with antibody followed by flow cytometry. Percent of TLR4-MD-2 + beads that were FITC-LPS + is shown. ( E-F ) Representative FACS plots (top) and quantification (bottom) showing the extent of LTA binding to TLR2-conjugated beads. (E) Human TLR2-conjugated beads were incubated with the peptides (48 or 96 μg/ml), washed, then incubated with LTA (1 μg/ml) and CD14 (0.1 μM). (F) CD14 (0.1 μM) was preincubated with equal molar of peptides (0.1 μM) before TLR2-conjugated beads and LTA (1 μg/ml) were added. In comparison, KAMP-10 alone (0.1 μM) was preincubated with TLR2-conjugated beads, followed by bead washing then incubation with LTA (1 μg/ml) and CD14 (0.1 μM). TLR2 and LTA were labeled with antibody for flow cytometric analysis. Percent of TLR2 + beads that were FITC-LTA + is shown. Mean (n=3 replicates) ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (ANOVA with Dunnett’s post hoc test).

Article Snippet: Similarly, to assess KAMPs binding to TLR2, TLR2-conjugated beads preincubated with individual KAMP at the aforementioned concentrations were washed with PBS, then incubated with S. aureus LTA (1 μg/ml) (Sigma-Aldrich) and recombinant human CD14 (R&D) (0.1 μM) for 1 h. To assess the effect of KAMPs on CD14-LTA interaction, KAMPs (0.1 μM equivalent to 78 ng/ml for KAMP-10 and SC-10; 160 ng/ml for KAMP-18C) were preincubated with equal molar of CD14 (0.1 μM) for 1 h on ice before incubation with TLR2-conjugated beads and LTA for 1 h. To detect LTA and TLR2 by flow cytometry, mouse monoclonal FITC anti-LTA antibody (clone 55; Novus Biologicals) and mouse recombinant PE anti-human/mouse TLR2 (clone QA16A01; Biolegend) were used.

Techniques: Binding Assay, Incubation, Labeling, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Cystin gene mutations cause autosomal recessive polycystic kidney disease associated with altered Myc expression

doi: 10.1101/2020.02.18.946285

Figure Lengend Snippet: Kidney histology and differential lectin staining. Formalin fixed, paraffin embedded 5 μm sections of kidney tissues from 6 week-old mice of the indicated genotypes were stained with H&E and examined by light microscopy ( A-F ) or stained with lectins LTA (green, proximal tubules) and DBA (red, distal tubules) and examined by immunofluorescence microscopy ( G,H,I ). Panels A-C are 5X magnification with scale bars equal to 100 μm. Boxed areas are shown at 40X magnification in corresponding panels D-F with scale bars equal to 20 μm. Lectin staining was performed on serial sections corresponding to H&E stained samples. Cell nuclei were stained with DAPI. Scale bars in panels G-I are equal to 10 μm. The asterisk (*) identifies a large cyst that was not stained by either LTA or DBA. Images are representative of tissue sections from 3 animals.

Article Snippet: Lectins LTA-FITC (# W0909) and DBA-Rhodamine (# Y0828) were obtained from Vector Laboratories.

Techniques: Staining, Formalin-fixed Paraffin-Embedded, Light Microscopy, Immunofluorescence, Microscopy