Goat anti mouse igg conjugated to horseradish peroxidase

To assess the ability of mannan to compete with LAM for binding to LprG, solid phase binding assays were performed as described [34] (link) in 96-well plates (Corning Incorporation, NY, USA) coated with ManLAM. ManLAM was added (100 ng in 50 µl of 50% ethanol per well), and plates were air-dried for 1 h at 37°C. Subsequent steps were at room temperature. Plates were washed in wash buffer (10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 1 mM CaCl₂, 0.005% Tween 20) to remove unbound ManLAM, blocked with 5% milk in wash buffer for 2 h, washed twice in wash buffer, and incubated overnight with 100 µl of NA-LprG (1 µM) that had been pre-incubated for 30 min with or without mannan (10–500 µM) in wash buffer minus Tween. The plates were washed extensively, incubated with monoclonal anti-LprG antibody (clone α-Rv1411c, NR13806, BEI) for 2 h, washed three times, incubated for 1 h with goat anti-mouse IgG conjugated to horseradish peroxidase (Cell Signaling Technology, Danvers, MA), washed three times and incubated with o-Phenylenediamine dihydrochloride (Sigma) in 0.05 M phosphate-citrate buffer, pH 5.0 (50 µl/well) in the dark for 30 min. To stop the color development reaction, 50 µl of 2N H₂SO₄ was added. OD₄₅₀ was determined on a Bio-Rad plate reader.

For immunoblotting the following primary (1) and secondary antibodies (2) were used: (1) caveolin 1 (anti-cav1 (D46G3) XP, mono-clonal, rabbit, Cell Signalling (Danvers, MA, USA), 1:1,000), EGFR (anti-EGFR (D38B1), mono-clonal, rabbit, Cell Signalling, 1:1,000), FGFRI (anti-FGFRI (Ab-154), poly-clonal, rabbit, Sigma, 1:200), FGFRII (anti-FGFRII (CD332), poly-clonal, rabbit, Thermo Scientific (Rockford, IL, USA), 1:1,000), PDGFRα (anti- PDGFRα (C-20), poly-clonal, rabbit, Santa Cruz (Dallas, TX, USA), 1:500), PDGFRβ (anti- PDGFRβ (958), poly-clonal, rabbit, Santa Cruz, 1:1,000), Pten (138G6), mono-clonal, rabbit, Cell Signalling, 1:1,000), Sfrp1 (anti-Sfrp1(EPR7003), mono-clonal, rabbit, Abcam (Cambridge, UK), 1:1,000), TGFβ1 (anti-TGFβ1 (ab9758), Abcam, poly-clonal, rabbit, 1:200), TGFβRI (anti- TGFβRI (H-100), poly-clonal, rabbit, Santa Cruz, 1:1,000), TGFβRII (anti- TGFβRII (D-2), mono-clonal, mouse, Santa Cruz, 1:1,000) and monoclonal mouse anti-β-Actin-Peroxidase (AC-15, Sigma, 1:10,000); (2) goat anti-rabbit and goat anti-mouse IgG conjugated to horseradish peroxidase (Cell Signalling, 1:10,000) as secondary antibody.