Journal: PLoS ONE

Article Title: Biologically Active Polymers from Spontaneous Carotenoid Oxidation: A New Frontier in Carotenoid Activity

doi: 10.1371/journal.pone.0111346

Figure Lengend Snippet: Balb/c mice were not supplemented (control) or supplemented daily by oral gavage with OxC-beta (10 mg/kg). After 4 weeks, intestinal tissues were harvested and CD14 and TLR-4 expression was determined by immunocytochemistry. Increased CD14 (A) and TLR-4 (C) expression is readily apparent in epithelial cells in the OxC-beta-supplemented animals compared to the controls receiving vehicle alone (B and D, respectively). Arrows indicate the location of enterocytes within the cross section of microvilli. Magnification 40x.

Article Snippet: Cells were stained for CD14, TLR-4, or TLR-2 by incubation for 1 hour at 4°C with the following phycoerythrin (PE) conjugated antibodies: anti-human CD14 (eBioscience 12–0149), anti-human TLR-4 (eBioscience 12–9917), and anti-human TLR-2 (eBioscience 12–9922).

Techniques: Expressing, Immunocytochemistry

Journal: International Journal of Molecular Sciences

Article Title: Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation

doi: 10.3390/ijms23095065

Figure Lengend Snippet: Specific recognition of C. acnes recombinant CAMP1 by TLR-2. ( A ) The recombinant proteins nmrCAMP1 (nm) and mrCAMP1 (m) (50 μg) were separated by electrophoresis in a 4–12% NuPAGE SDS BisTris gel and transferred onto nitrocellulose membranes, which were incubated with recombinant TLR-2, TLR-1, TLR-4 and TLR-6 (0.1 μg/mL). TLR binding activity was detected with specific biotinylated antibodies against TLR-2, TLR-1, TLR-4 and TLR-6, respectively, as described in the Materials and Methods. The arrow indicates the position of the 47 kDa band of interest. ( B ) The recombinant proteins were separated by electrophoresis in a 4–12% NuPAGE SDS BisTris gel (50 μg) with detection by Coomassie brilliant blue staining. The separated proteins were transferred onto nitrocellulose membranes, which were incubated with poly-histidine antibody or recombinant TLR-2 (0.1 μg/mL), with the detection of TLR binding activity with specific biotinylated antibodies against TLR-2. Lanes 1a and 1b contain the molecular mass markers. Arrows indicate the positions of the 30 and 47 kDa bands of interest. The peptide sequences of the 47 kDa nmrCAMP1 and mrCAMP1 and the 30 kDa protein were obtained by LC-MS/MS (in bold, the amino acid substitutions in mrCAMP1). ( C ) Immobilized nmrCAMP1 (square) and mrCAMP1 (circle) (20 μg/mL) were probed with various concentrations of TLR-1, TLR-2, TLR-4 or TLR-6 (0.016 to 16 μg/mL).

Article Snippet: The plates were washed three times with PBS-Tween, and biotinylated human antibodies against TLR-1, TLR-2, TLR-4 or TLR-6 (R&D System, Abingdon, UK) diluted to 0.05 μg/mL in PBS-Tween were added and the plates were incubated at room temperature for 1 h. The plates were washed, peroxidase-conjugated streptavidin (0.5 μg/mL in PBS-Tween) was added and the plates were incubated for 30 min at room temperature.

Techniques: Recombinant, Electrophoresis, Incubation, Binding Assay, Activity Assay, Staining, Liquid Chromatography with Mass Spectroscopy