biotinylated human antibodies against tlr 2  (R&D Systems)

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: Expression of Christie–Atkins–Munch–Petersen (CAMP)1 in Cutibacterium acnes ( C. acnes ) strains. C. acnes surface proteins were extracted from a five-day culture ( A , C ) in liquid RCM medium or ( B ) on solid RCM medium and separated by electrophoresis in a 4–12% NuPAGE SDS BisTris gel (50 μg). Separated proteins were transferred onto nitrocellulose membranes, which were incubated ( A , B ) with antibodies against CAMP1 or ( C ) with recombinant toll like receptor (TLR)-2. TLR-2 binding activity was detected with specific biotinylated antibodies against TLR-2, as described in the Materials and Methods. TLR-2 binding activity previously reported by Lheure et al. . Lanes 1 to 12 correspond to phylotype IA1 strains: 75150, 16351, 17248, 53468, 41103, 6919, 78910, 36862, RON, 22197, LIE and 31430, respectively. Lane 13 corresponds to phylotype IA2 strain: CHR. Lanes 14 to 22 correspond to phylotype IB strains: 14230, 12513, 22795, 47474, 27387, 25236, GUE, PIE and 54137, respectively. Lane 23 corresponds to phylotype II strain: 27647. ( D ) HaCaT cells were stimulated for 1, 4, 6 and 24 h at 37 °C, with C. acnes strains 6919 (phylotype IA1), 14230 (phylotype IB) or PIE (phylotype IB) grown in liquid or on solid medium. C-X-C motif chemokine ligand interleukin (CXCL)8/(IL)-8 protein levels were assessed by enzyme-like immunosorbent assay (ELISA). nd: non-determined.

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: Expressing, Electrophoresis, Incubation, Recombinant, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

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: Inhibition of C. acnes -induced CXCL8/IL-8 production by TLR-2 and CAMP1 antibodies. HaCaT cells were treated with ( A ) anti-TLR-2 antibody (1 μg/mL) or ( B ) anti-CAMP1 antibody (1 μg/mL) for 24 h (gray bar), and were then stimulated with various strains of C. acnes (multiplicy of infection 15 (MOI 15) (phylotype IA1, strain 6919; phylotype IB, strains PIE and 14230) for 18 h (dark bar). Control experiments correspond to C. acnes stimulation of HaCaT cells only (gray bar). CXCL8/IL-8 production was measured by ELISA. Statistical significance is indicated by ** ( p < 0.01), *** ( p < 0.001) and **** ( p < 0.0001).

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: Inhibition, Infection, Enzyme-linked Immunosorbent Assay

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: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

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

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: Subpeptides from CAMP1 recognized by TLR-2. ( A , B , C ) The peptides derived from CAMP1 proteins: group F (positions A1-C21), group A (positions C22-E9), group C (positions E10-E13), group D (positions E14-E17), group E (positions E18-E20); a biotinylated peptide used as a positive control (position F22) and a peptide used as a negative control (position F24), were immobilized on glass plates. ( A ) The plates were incubated with TLR-2 (10 μg/mL)/anti-TLR-2 antibody (0.1 μg/mL)/HRP (0.5 μg/mL) and ( B ) with anti-TLR-2 antibody (0.1 μg/mL)/HRP (0.5 µg/mL). The arrows indicate the positions of peptides specifically recognized by TLR-2. ( D ) Immobilized rCAMP1 (20 μg/mL) proteins were probed with biotinylated TRL-2 (0.5 μg/mL) (dark bar) after pre-treatment with peptide (A14, A15, B1, B2, C1 and C3) at various concentrations (1.25, 12.5, 125, 1250 and 12,500 nM). Statistical significance is indicated by * ( p < 0.05), ** ( p < 0.01) and *** ( p < 0.001).

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: Derivative Assay, Positive Control, Negative Control, Incubation

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: In silico 3D analysis of C. acnes factor CAMP1. ( A ) Sequence alignment of CAMP factors from different species. Conservatively substituted residues are boxed and strictly conserved residues are highlighted with a red background. The figure was constructed with ESPript 3.0. Reference sequences: Cutibacterium acnes nmCAMP1: AAS92206.1; Cutibacterium acnes mCAMP1: KX581410; Streptococcus agalactiae : ZP_08649639.1; Mobiluncus curtisii : YP_003718285.1; Streptococcus pyogenes : WP_111681137.1. ( B – D ) Predicted structures of the nmCAMP1 and mCAMP1 proteins obtained by homology modeling with the trRosetta server and the images generated with PyMOL software. ( B ) Cartoon representation of CAMP1. The NTD domain is colored in yellow, the CTD domain in cyan and the linker region in red. ( C ) CAMP1 peptides involved in TLR-2 binding are highlighted: A14 in blue, A15 in cyan, B1 in yellow, B2 in orange, B3 in red, C1 in purple and C3 in pink. ( D ) Surface representation of CAMP1 peptides.

Article Snippet: Unbound antibodies were removed by washing, as described above, and slides were incubated with biotinylated human antibodies against TLR-2 (R&D Systems, Abingdon, UK) diluted to 0.05 μg/mL in TBS/T supplemented with 5% BSA, for 20 h at 4 °C, with gentle mixing.

Techniques: In Silico, Sequencing, Construct, Generated, Software, Binding Assay