Mouse Anti-F2RL3 Recombinant Antibody (clone P20.1); Fab Fragment (CAT#: PFBC-043)

Figure 1 Flow cytometric analysis of FLAG-PAR4 expressing cells.

FLAG-PAR4/T-REx293 cells treated with (shaded histogram) or without (solid line histogram) tetracycline were stained with an anti-FLAG M1 (upper panel) or P20.1 (lower panel) and FITC-conjugated secondary antibody. Dotted line histograms are stained with parental T-REx 293 cells.

Sangawa, T., Nogi, T., & Takagi, J. (2008). A murine monoclonal antibody that binds N-terminal extracellular segment of human protease-activated receptor-4. Hybridoma, 27(5), 331-335.

Figure 2 Sensorgrams from the kinetic analyses of the binding between P20.1 IgG and immobilized P4-Fn (A), or anti-FLAG M2 IgG and immobilized FLAG-Fn (B).

Serially diluted antibodies at the indicated concentrations were applied onto the surface immobilized with the respective Fn fusion proteins. All data are after subtraction of signals from control surface (FLAG-Fn immobilized surface for P20.1 and P4-Fn surface for M2). Arrows indicate start and endpoints of the injection.

Sangawa, T., Nogi, T., & Takagi, J. (2008). A murine monoclonal antibody that binds N-terminal extracellular segment of human protease-activated receptor-4. Hybridoma, 27(5), 331-335.

Figure 3 Enzyme-linked immunosorbent assay (ELISA) experiments presenting the binding of P20.1 to various PAR4 peptides.

(A) Effect of truncation. Fusion proteins bearing various lengths of PAR4 peptide were coated on microtiter wells and incubated with P20.1 hybridoma culture supernatant, followed by detection with anti-mouse IgG-HRP. Bindings are expressed as the relative absorbance value normalized to the full-length control (i.e., P4-Fn). (B) Alanine scanning. Fusion proteins bearing either wildtype (WT) or alanine-mutagenized PAR4 peptide were coated on microtiter wells and incubated with increasing concentrations (0.003–3 μg/mL) of P20.1 IgG. Data are after subtraction of absorbance obtained with control wells that had been coated with BSA only.

Sangawa, T., Nogi, T., & Takagi, J. (2008). A murine monoclonal antibody that binds N-terminal extracellular segment of human protease-activated receptor-4. Hybridoma, 27(5), 331-335.

Figure 4 P20.1 inhibits PAR4 peptide cleavage by thrombin.

Inhibition of thrombin cleavage. P4-Fn (4.4 μM) was treated without (lane 1) or with (lanes 2–12) 27 nM thrombin for 1 h. Prior to this treatment, P20.1 IgG (lanes 3–7) or Fab fragment (lanes 8–12) were added at the following concentrations and preincubated for 2 h. Lane 3, 0.55 μM; lane 4, 1.1 μM; lane 5, 2.2 μM; lane 6, 4.4 μM; lane 7, 8.8 μM; lane 8, 1.1 μM; lane 9, 2.2 μM; lane 10, 4.4 μM; lane 11, 8.8 μM; lane 12, 17.6 μM. Note that the IgG concentrations must be doubled to make them Fab equivalent.

Sangawa, T., Nogi, T., & Takagi, J. (2008). A murine monoclonal antibody that binds N-terminal extracellular segment of human protease-activated receptor-4. Hybridoma, 27(5), 331-335.